WO2005111627A2 - Procedes et produits associes a l'analyse amelioree de glucides - Google Patents

Procedes et produits associes a l'analyse amelioree de glucides Download PDF

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Publication number
WO2005111627A2
WO2005111627A2 PCT/US2005/013107 US2005013107W WO2005111627A2 WO 2005111627 A2 WO2005111627 A2 WO 2005111627A2 US 2005013107 W US2005013107 W US 2005013107W WO 2005111627 A2 WO2005111627 A2 WO 2005111627A2
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Prior art keywords
glycans
sample
glycoconjugates
analyzing
glycan
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PCT/US2005/013107
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WO2005111627A3 (fr
Inventor
Nishla Keiser
Carlos Bosques
Ram Sasisekharan
Pankaj Gandhe
S. Raguram
Aravind Srinivasan
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Massachusetts Institute Of Technology
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Publication of WO2005111627A3 publication Critical patent/WO2005111627A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Definitions

  • the invention relates to the improved analysis of carbohydrates.
  • the invention relates to the analysis of carbohydrates, such as N-glycans and O-glycans found on proteins and lipids.
  • the invention also relates to the analysis of glycoconjugates, such as glycoproteins, glycolipids and proteoglycans.
  • Methods for the study of glycosylation patterns on cells, tissues and in body fluid, such as serum, are also provided.
  • Information regarding the glycosylation patterns on cells can be used in diagnostic and treatment methods as well as for facilitating the study of the effects of glycosylation/altered glycosylation on diseases, protein or lipid function and function of medical treatments.
  • Information regarding the glycosylation of glycoconjugates can also be used in the quality control analysis of glycoconjugate production and/or therapeutics.
  • Asparagine-linked glycosylation is the most common co- translational modification found in eukaryotic proteins. As proteins are synthesized by the ribosome, the polypeptide enters the endoplasmic reticulum, where oligosaccharyl transferase (OT) attaches a branched carbohydrate (N-glycan) to the side chain of certain asparagine residues [ Hirschberg, C.B., Snider, M.D. (1987) Topography of glycosylation in the rough endoplasmic reticulum and Golgi apparatus.
  • OT oligosaccharyl transferase
  • N- glycans are very important in protein folding, as well as directing the protein to the appropriate location within the cell [ Dwek, R.A. (1996) Glycobiology: Toward Understanding the Function of Sugars. Chem Rev 96, 683-720; O'Connor, S.E., Imperiali, B. (1996) Modulation of protein structure and function by asparagine-linked glycosylation. Chem Biol 3, 803-12.] Outside the cell, the sugars aid in protein-protein interactions, often modulating the activity of the protein to which they are attached. Depending on the glycan composition, they can also protect against or facilitate protein degradation in circulation, as well as target the protein to a specific organ [Crocker, P.R., Varki, A.
  • N-glycans also have an essential role in normal biology, as evidenced by the high lethality in cases of defective glycosylation.
  • selectins a family of proteins expressed on endothelial cells or lymphocytes that can trigger the immune system upon activation
  • selectins a family of proteins expressed on endothelial cells or lymphocytes that can trigger the immune system upon activation
  • Natural ligands of the B cell adhesion molecule CD22 beta carry N-linked oligosaccharides with alpha-2,6-linked sialic acids that are required for recognition. JBiol Chem 268, 7019-27; Sgroi, D., Varki, A., Braesch-Andersen, S., Stamenkovic, I.
  • CD22 a B cell-specific immunoglobulin superfamily member, is a sialic acid-binding lectin. JBiol Chem 268, 7011-8.]
  • the same class of structures that are necessary for proper immune, function can also provide a binding site for certain viruses, bacteria or tumor cells in the body [Karlsson, K.A. (1998) Meaning and therapeutic potential of microbial recognition of host glycoconjugates. Mol Microbiol 29, 1-11; Pritchett, T.J., Brossmer, R., Rose, U., Paulson, J.C. (1987) Recognition of monovalent sialosides by influenza virus H3 hemagglutinin.
  • Viral infection is mediated by the interaction of viral proteins with N-glycans on the cell surfaces of the host [ Van Eijk, M., White, M.R., Batenburg, J.J., Vaandrager, A.B., Van Golde, L.M., Haagsman, H.P., Hartshorn, K.L. (2003) Interactions of Influenza A virus with Sialic Acids present on Porcine Surfactant Protein D. Am JRespir Cell Mol Biol] Despite the increasing evidence associating glycans to different pathogenic conditions, in multiple instances it is unclear whether changes in N-glycan structure are a cause or a symptom of the disorder.
  • cystic fibrosis increased antennary fucosylation ( ⁇ l-3 linked to GlcNAc) is observed on surface membrane glycoproteins of airway epithelial cells [ Glick, M.C., Kothari, V.A., Liu, A., Stoykova, L.I., Scanlin, T.F. (2001) Activity of fucosyltransferases and altered glycosylation in cystic fibrosis airway epithelial cells. Biochimie 83, 743-7; Scanlin, T.F., Glick, M.C. (2000) Terminal glycosylation and disease: influence on cancer and cystic fibrosis.
  • Glycobiology 10, 173-6. Melanoma and bladder cancer cells produce proteins with highly branched glycans due to an overexpression of the biosynthetic enzyme ⁇ l,6-N-acetyl-glucosaminyltransferase V (GnT-V) [Chakraborty, A.K., Pawelek, J., Ikeda, Y., Miyoshi, E., Kolesnikova, ⁇ ., Funasaka, Y., Ichihashi, M., Taniguchi, ⁇ .
  • This invention provides, in part, methods related to the analysis of carbohydrates.
  • the invention relates to the analysis of carbohydrates, such as N-glycans and O- glycans found on proteins and lipids.
  • the invention also relates to the analysis of glycoconjugates, such as glycoproteins, glycolipids and proteoglycans.
  • methods of analyzing a sample containing glycoconjugates include (a) separating the carbohydrates (e.g., glycans) from the sample containing the glycoconjugates, (b) determining the glycosylation site and/or occupancy of the glycoconjugates, (c) analyzing the carbohydrates (e.g., glycans) for characterization and/or quantification, and (d) determining the glycoforms and/or glycan profile of the glycoconjugates in the sample with the results obtained from steps (b) and (c) with a computational approach.
  • the methods also include determining the occupancy of each glycan at each glycosylation site.
  • step (a) includes denaturing the glycoconjugates.
  • the glycoconjugates are denatured with a denaturing agent.
  • the denaturing agent is detergent, urea, guanidium hydrochloride or heat.
  • the glycoconjugates are reduced following their denaturation.
  • the glycoconjugates are reduced with a reducing agent.
  • the reducing agent in certain embodiments is DTT, ⁇ -mercaptoethanol or TCEP.
  • the glycoconjugates are alkylated with an alkylating agent following their reduction.
  • the alkylating agent in certain embodiments is iodoacetic acid or iodoacetamide.
  • the step of determining the glycosylation sites and/or glycosylation site occupancy includes analyzing the glycoconjugates, preferably with 2D-NMR.
  • the step of determining the glycosylation site and glycosylation site occupancy includes cleaving the peptide backbone of the glycoconjugates (and optionally analyzing the cleaved fragments), cleaving and labeling with a first label the glycoconjugates at their glycosylation sites of a first portion of the sample, cleaving the glycoconjugates at their glycosylation sites of a second portion of the sample, analyzing the first and second portions of the sample of glycoconjugates, and quantifying the results.
  • the analysis of the first and second portions of the sample can be performed separately or mixed in any ratio.
  • the glycoconjugates of the first portion are labeled with a label.
  • the label is an isotope of C, N, H, S or O. More preferably, the label is O 18 .
  • the glycoconjugates of the second portion are unlabeled.
  • the glycoconjugates of the second portion are labeled.
  • the first and second portions of the sample of glycoconjugates are analyzed separately.
  • the first and second portions of the sample of glycoconjugates are analyzed as a mixture.
  • the glycosylation site occupancy is quantified from ratios of the masses of cleaved glycoconjugates of the first and second portions of the sample.
  • the first and second portions of the sample are analyzed with a mass spectrometric method.
  • the mass spectrometric method is LC-MS, LC- MS/-MS, MALDI-MS, MALDI-TOF, TANDEM-MS or FTMS.
  • the step further includes generating a list of possible glycoconjugates and/or and peptides, e.g., using databases.
  • the step further includes generating a list of possible glycans.
  • the step of analyzing the glycans includes, in certain embodiments, analyzing the glycans with a mass spectrometric method, an electrophoretic method, NMR, a chromatographic method or a combination thereof.
  • the mass spectrometric method is LC-MS, LC-MS/MS, MALDI-MS, MALDI-TOF, TANDEM-MS or FTMS.
  • the mass spectrometric method is a quantitiative MALDI-MS or MALDI- FTMS using optimized conditions.
  • the MALDI-MS is MALDI- MS optimized with a mixture of 6-aza-2-thiothymine (ATT) and Nafion ® coating.
  • the electrophoretic method is CE-LIF.
  • the step further includes contacting the glycans with one or more glycan-degrading enzymes.
  • the one or more glycan- degrading enzymes is sialidase, galactosidase, mannosidase, N-acetylglucosaminidase or a combination thereof.
  • the step of analyzing the glycans includes quantifying the glycans using calibration curves of known glycan standards.
  • the method further includes determining a peptide sequence of the glycoconjugate.
  • low abundance species are detected due to the low detection limits, which preferably extend to lower than about 5 finol.
  • Low abundance species include, but are not limited to, fucoses, sialic acids, galactoses, mannoses and sulfate groups.
  • methods of analyzing a sample containing glycoconjugates include separating glycans from the sample containing the glycoconjugates, determining the glycosylation sites and glycosylation site occupancy of the glycoconjugates, and analyzing the glycans to characterize and/or quantify the glycans.
  • Determining the glycosylation sites and glycosylation site occupancy includes cleaving and labeling with a first label the glycoconjugates of a first portion of the sample at their glycosylation sites, cleaving the glycoconjugates of a second portion of the sample at their glycosylation sites, analyzing the first and second portions of the sample of glycoconjugates, and quantifying the results.
  • the glycoconjugates of the first portion are labeled.
  • the glycoconjugates of the second portion are unlabeled.
  • the glycoconjugates of the second portion are labeled.
  • the first and second portions of the sample of glycoconjugates are analyzed with a mass spectrometric method.
  • the mass spectrometric method is LC-MS, LC- MS/MS, MALDI-MS, MALDI-TOF, TANDEM-MS or FTMS.
  • determining the glycosylation sites and glycosylation site occupancy further includes generating a list of possible glycoconjugates.
  • the step of separating the glycans from the sample includes denaturing the glycoconjugates with a denaturing agent.
  • the glycoconjugates are reduced with a reducing agent following their denaturation. More preferably, the glycoconjugates are alkylated with an alkylating agent following their reduction.
  • the step of analyzing the glycans includes analyzing the glycans with a mass spectrometric method, an electrophoretic method, NMR, a chromatographic method or a combination thereof.
  • the mass spectrometric method preferably is LC-MS, LC-MS/MS, MALDI-MS, MALDI-TOF, TANDEM-MS or FTMS.
  • the electrophoretic method preferably is CE-LIF.
  • the step further includes contacting the glycans with one or more glycan-degrading enzymes.
  • the one or more glycan-degrading enzymes is sialidase, galactosidase, mannosidase, N-acetylglucosaminidase or a combination thereof.
  • the method further includes determining a peptide sequence of the glycoconjugate. According to still another aspect of the invention, methods of determining the glycosylation site occupancy of glycoconjugates in a sample are provided.
  • the methods include cleaving and labeling with a first label the glycoconjugates at their glycosylation sites of a first portion of the sample, cleaving the glycoconjugates at their glycosylation sites of a second portion of the sample, analyzing the first and second portions of the sample of glycoconjugates, and quantifying the results.
  • the method further includes determining the possible fragments of the glycoconjugate.
  • the glycoconjugates of the first portion are labeled with an isotope of C, N, H, S or O. Preferably the label is O 18 .
  • the glycoconjugates of the second portion are unlabeled.
  • the glycoconjugates of the second portion are labeled.
  • the first and second portions of the sample of glycoconjugates are analyzed with a mass spectrometric method.
  • the mass spectrometric method is LC-MS, LC- MS/MS, MALDI-MS, MALDI-TOF, TANDEM-MS or FTMS.
  • low abundance species are detected due to the low detection limits, which preferably extend to lower than about 5 fmol. Low abundance species include, but are not limited to, fucoses, sialic acids, galactoses, mannoses and sulfate groups.
  • methods of analyzing a sample containing glycans include separating neutral from charged glycans, and analyzing the neutral and charged glycans separately to analyze the glycan.
  • the analysis of the glycans is performed with MALDI-MS.
  • methods of analyzing a glycan are provided. The methods include analyzing the glycan in the presence of National ® and 6-aza-2-thiothymine (ATT). Certain embodiments of the foregoing methods are methods of analyzing the purity of a sample containing glycans.
  • inventions of the foregoing methods are methods of analyzing the glycans of a sample of a cell, a group of cells, a tissue or serum or other body fluid from a subject. Still other embodiments of the foregoing methods are high-throughput methods, in which more than one sample of glycoconjugates is analyzed. In some preferred embodiments, the more than one sample of glycoconjugates are in a 96-well plate. In other preferred embodiments, the more than one sample of glycoconjugates are on a membrane.
  • carbohydrate cleavage is performed using enzymes such as PNGase F, endoglycosydase H, or endoglycosydase F, or chemical methods such as hydrazinolisis or alkali borohydrate cleavage.
  • cleavage is performed in a high-throughput manner in 96-weU plates or in solution.
  • purification is performed using solid phase extraction cartridges such as graphitized carbon columns and C-18 columns.
  • purification is performed in a high-throughput manner in 96-well plates. All of the foregoing steps, particularly the step of separation, can be performed with the use of robotics. - According to another aspect of the invention, methods of generating a glycoconjugate,
  • glycopeptide library preferably glycopeptide library
  • the methods include cleaving the backbone of the glycoconjugate (preferably the peptides of the glycopeptides) in a sample and labeling the fragments generated with a first labeling agent, and cleaving the glycans in the sample and labeling the fragments generated in the sample with a second labeling agent.
  • the library represents all possible glycoform fragments of the sample containing the glycoconjugates.
  • the glycoconjugates preferably are glycopeptides, glycolipids or proteoglycans.
  • the first and second labeling agent is the same labeling agent.
  • the labeling agent is an isotope of C, N, H, S or O, preferably O 18 .
  • the method further includes characterizing the fragments generated from the cleavage of the glycopeptides.
  • the characterization is performed with LC-MS, LC-MS/MS, MALDI-MS, MALDI-TOF, TANDEM-MS or FTMS.
  • the characterizing includes characterizing the glycosylation sites, characterizing the peptides of the glycopeptides and/or characterizing the glycans.
  • a library of glycopeptides generated with the foregoing methods is generated. The library can be used as an internal standard to analyze new batches of glycoconjugates by direct comparison to each labeled standard from the library.
  • the backbones of new batches of glycoconjugates can be cleaved and mixed with the labeled fragments from the library to characterize all the glycoforms present in the new batch from the ratios of labeled and unlabeled fragments.
  • methods of analyzing a sample of glycopeptides include analyzing the glycopeptides, and comparing the analyzed glycopeptides with the foregoing library of glycopeptides of the foregoing embodiments. In certain embodiments, it is preferred that comparative characterization is performed using LC-MS, LC-MS/MS, MALDI-MS, MALDI-TOF, TANDEM-MS or FTMS.
  • methods of generating a list of glycoconjugate properties include measuring two or more properties of the glycoconjugate, and recording a value for the two or more properties of the glycoconjugate to generate a list, wherein the value of the two or more properties is recorded in a computer-generated data structure.
  • one of the two or more properties of the glycoconjugates is the number of one or more types of monosaccharides of __ the glycoconjugate.
  • one of the two or more properties of the glycoconjugates is the total mass of the glycans of the glycoconjugate.
  • the glycoconjugate is a glycoprotein or proteoglycan, and one of the two or more properties of the glycoconjugate is the mass of the peptide of the glycoconjugate.
  • the glycoconjugate is a glycolipid, and one of the two or more properties of the glycoconjugate is the mass of the lipid of the glycoconjugate.
  • one of the two or more properties of the glycoconjugate is the mass of the glycoconjugate.
  • one of the two or more properties of the glycoconjugate is the mass of permethylated glycans.
  • a database tangibly embodied in a computer- readable medium, for storing information descriptive of one or more glycoconjugates.
  • the database includes one or more data units corresponding to the one or more glycoconjugates, each of the data units including an identifier that includes two or more fields, each field for storing a value corresponding to one or more properties of the glycoconjugates.
  • methods of analyzing the total glycome of a sample of body fluid, cells or tissues are provided. The methods include (a) analyzing all the glycans of the sample, and (b) determining a profile of the glycans of the sample.
  • the sample is optionally fractionated and/or the glycans are separated from the glycoconjugates.
  • the cleavage, fractionation, purification and/or separation steps described elsewhere herein are optionally included in the methods.
  • the method further includes performing a pattern analysis on the results from (a) using computational tools.
  • the pattern can be described as (but is not limited to) relative amounts of the components of the pattern, absolute amounts of the components of the pattern, ratios between the components of the pattern, combinations of different components of the pattern, presence or absence of any of the components of the pattern or combination of the above.
  • the identification of the glycome pattern and the pattern analysis can be performed using computational methods.
  • this includes an iterative process, which optionally includes one of more of the following: incoiporation of all experimental data sets from the glycome analysis and other glycan characterization, generation of theoretical glycan structures, incorporation of glycan composition, incorporation of structure and property information from databases, incorporation of glycan biosynthetic pathway information, incorporation of patient (or sample origin) information such as patient history and demographics, extract features from the experimental data sets, generation of data sets with specific features, submitting the combined information to data mining analysis, establishing relationship rules and validating the patterns.
  • step (a) includes quantifying the glycans using calibration curves of known glycan standards.
  • the method further includes recording the pattern in a computer-generated data structure.
  • the method is a method for diagnostic or prognostic purposes.
  • the method is a method for assessing the purity of the sample.
  • the sample is a sample of serum, plasma, blood, urine, saliva, sputum, tears, CSF, seminal fluid, feces, tissues or cells.
  • methods of analysis are provided. The methods include (a) analyzing all of the glycans of a sample of body fluid, cells and/or tissues and (b) comparing the results from (a) with a known pattern.
  • the sample is a sample of serum, plasma, blood, urine, saliva, sputum, tears, CSF, seminal fluid, feces, tissues or cells.
  • the methods are methods of diagnosis and the pattern is associated with a diseased state.
  • the pattern associated with a diseased state is a pattern associated with cancer, such as prostate cancer, melanoma, bladder cancer, breast cancer, lymphoma, ovarian cancer, lung cancer, colorectal cancer or head and neck cancer.
  • the pattern associated with a diseased state is a pattern associated with an immunological disorder; a neurodegenerative disease, such as a transmissible spongiform encephalopathy, Alzheimer's disease or neuropathy; inflammation; rheumatoid arthritis; cystic fibrosis; or an infection, preferably viral or bacterial infection.
  • the method is a method of monitoring prognosis and the known pattern is associated with the prognosis of a disease.
  • the method is a method of monitoring drug treatment and the known pattern is associated with the drug treatment.
  • the methods are used for the selection of population-oriented drug treatments and/or in prospective studies for selection of dosing, for activity monitoring and/or for determining efficacy endpoints.
  • methods of determining the purity of a sample include (a) analyzing total glycans of the sample, (b) identifying the glycan pattern of the sample, and (c) comparing the pattern with a known pattern to assess the purity of the sample. Similar methods are provided in which glycoconjugates in sample are analyzed.
  • a method of generating the complete glycan pattern of a body fluid, cells and/or tissue is provided.
  • the method includes, (a) analyzing the glycans in a sample of the body fluid, cells and/or tissue, and (b) identifying the complete glycan pattern of the sample.
  • neutral, charged, N-linked and O-linked glycans are included in the pattern.
  • glycosaminoglycans and glycolipids are included in the pattern.
  • the sample is a sample of serum, plasma, blood, urine, saliva, sputum, tears, CSF, seminal fluid, feces, tissues or cells.
  • methods of analyzing the total glycome of a sample are provided.
  • the methods include determining the glycosylation site and glycosylation site occupancy of all glycoconjugates in the sample, characterizing components of the glycoconjugates and all glycans of the glycome in the sample, and matching specific glycans to glycoconjugates with a computational method.
  • methods of analyzing a sample of glycoconjugates are provided. The methods include analyzing the glycans of the sample with an analytical method, and determining the glycoforms of the sample with a computational method. In certain embodiments of the foregoing methods, the methods include generating constraints from the experimental analysis and solving them.
  • a further method for matching each carbohydrate in the glycome to its glycoconjugate includes characterization of glycosylation sites and occupancy of all glycoconjugates from body fluids, determination of possible glycans at each site by comparing unlabeled, glycoconjugate fragments to labeled, deglycosylated fragments, characterization of the entire glycome from body fluids, and combination of the different datasets into the iterative computational analysis to match the glycans to the glycoconjugates.
  • Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention.
  • FIG. 1 shows the conserved N-glycan pentasaccharide core.
  • Figr2 illustrates classes of N-linked glycans. High-mannose structures contain-up to
  • Fig. 2A Complex type glycans are modified with hexosamines, galactoses, sialic acids and/or fucose, among other residues (Fig. 2B). Complex type chains can occur as mono-, bi-, tri-, and tetra-antennary structures. Also, the amount and type of sialylation differs. Hybrid structures contain characteristics of both high-mannose and complex types (Fig.2C).
  • Fig. 3 provides the detailed pathway of N-glycan biosynthesis (http://www.genome.ad.jp/kegg/pathway/map/map00510.html).
  • Fig. 4 shows the cleavage sites of EndoH, EndoF and PNGaseF.
  • Fig. 5 provides the MALDI-MS spectra of N-glycans from RNaseB samples prepared by various methods. Glycans after GlycoClean S (Table 2, Sample 12), with the expected high mannose peaks and significant contamination of unknown identity (Fig. 5A). A small amount of sample (10 ⁇ g) was prepared using a 25 mg GlycoClean H column (Table 2, Sample 17), which showed only detergent peaks (Fig.5B).
  • Fig. 5C A larger amount of protein (50 ⁇ g) was prepared (Table 2, Sample 18), yielding the expected glycan peaks but still containing detergent contamination (Fig. 5C).
  • Fig. 5D Using a 200 mg GlycoClean H column to purify N-glycans from 150 ⁇ g of RNaseB (Table 2, Sample 20), only the high mannose saccharides were observed (Fig. 5D).
  • Fig. 6 shows the spectra from MALDI-MS of N-glycans from ovalbumin. Each labeled peak corresponds to a previously reported structure listed in Table 3.
  • Fig. 7 provides results from a study of N-glycans from antibody samples. Figs.
  • Figs. 7C-7E are for samples from Wave reactors.
  • Fig. 7C represents the results for DO controlled, pH uncontrolled, and NaOH in the media
  • Fig. 7D represents the results with NaHCO 3 in the media instead of NaOH.
  • Fig. 8 shows the structures and theoretical masses of N-glycans released from antibodies.
  • Fig. 9 MALDI-MS spectra of glycans released from serum proteins using PNGaseF and EndoF. Serum samples were treated with PNGaseF (Fig.
  • FIG. 10 shows a separation of neutral and acidic glycans using GlycoClean H cartridge, (a) The original mix of standards is shown in positive mode. A3 and SCI 840 are additional highly charged, and do not ionize well, (b) Neutral glycans eluted off the GlycoH cartridge ionize well in positive mode, while only the charged sugars are present in (c), allowing them to be observed in negative mode. The multiple peaks in (c) arise from sodium adducts, typically one adduct per sialic acid residue.
  • Fig. 11 provides the results from MALDI-MS of N-glycans from human serum in neutral (left) and acidic (right) fractions, (a) and (b) represent neutral glycans prepared from two different IMPATH normal male human serum samples, while (d) and (e) show the acidic fraction, (c) and (f) are the neutral and acidic fractions of a normal human sample from Biomedical Resources.
  • Fig. 12 provides the results of serum glycans separated by ConA. (a) SDS-PAGE of CoiiA flow through (Lane 2) and elution (Lane 3). vLane 1 shows molecular weight standards.
  • vMALDI-MS of (b) neutral and (c) acidic sugars obtained from ConA elution Fig. 13 provides the results from protein A separation of IgG from serum, (a) Glycoblot of Protein A flow-through (Lane 3) and elution (Lane 4). Lane 1 contains protein standards, while Lane 2 (negative control) contains human serum albumin (* marks where albumin would run on an SDS-PAGE gel). Only glycosylated proteins are observed in the glycoblot, so the albumin does not stain.
  • MALDI-MS of glycans harvested from the elution fraction are shown in (b) neutral and (c) acidic. Total serum glycans are pictured in (d) neutral and (e) acidic.
  • Fig. 14 shows the permethylation of N-glycans. All OH and ⁇ H groups can be permethylated. For complete reaction, it is essential that the reaction vessel is free of air and water.
  • Fig. 15 shows the results of MALDI-MS of permethylated glycan standards, (a) Unmodified standards ionized unevenly. Permethylated standards (b) showed more uniform ionization, but generally did not have higher signal-to-noise ratios.
  • Fig. 16 shows the aminooxyacetyl peptide and its conjugation to N-glycans. The aminooxyacetate end of the synthetic peptide (top) reacts with the open form of the reducing end Glc ⁇ Ac of N-glycans (bottom).
  • Fig. 17 shows the results of MALDI-MS of peptide-conjugated N-linked standards,
  • Fig. 18 shows the identification of serum N-glycans from MALDI-MS spectra, (a) shows neutral glycans, while (b) shows acidic glycans. Labeled peak numbers correspond to entries in Table 7.
  • Fig. 19 shows the results of neutral N-glycans from PVDF digest. Only the most abundant glycans are observed.
  • Fig. 20 provides a MALDI spectra of glycans before (A) and after (B) applying new recipe with optimized conditions. Fig.
  • Fig. 21 provides results from glycan quantification using optimized matrix recipe for MALDI-MS
  • Fig. 22 provides a schematic of an example of a methodology for analysis.
  • Fig. 23 provides a flowchart illustration of one example of a combined analytical- computational method for glycan analysis.
  • Fig. 24 provides a scheme for an exemplary method for glycoprotein analysis - glycan site occupancy analysis.
  • Fig. 25 provides results from glycan site occupancy analysis for ribonuclease B.
  • the expected [M+H]+ for the unlabeled peptide fragment is 476.29 Da.
  • Fig. 26 provides MALDI-MS spectra of N-glycans from RNaseB with the expected high mannose structures.
  • Fig. 27 provides results from MALDI-MS of N-glycans from ovalbumin. Each labeled peak corresponds to a previously reported structure listed below.
  • Fig. 28 provides structures and theoretical masses of N-glycans released from antibodies.
  • Fig. 29 shows the results from an analysis of depletion of serum albumin and IgGs from serum.
  • Fig. 30 shows the results of protein A separation of IgG from serum, (a) Glycoblo
  • Fig. 31 shows the identification of serum N-glycans from MALDI-MS spectra, (a) shows neutral glycans, while (b) shows acidic glycans.
  • Fig. 32 provides the results from LC-MS (A) and CE-LIF(B) analysis of neutral glycome from serum.
  • Fig. 33 provides the MALDI-MS acidic glycome profile of saliva (A) and urine (B).
  • Fig. 34 provides quantitative neutral glycome profile for serum with normal (A) and low (B) IgG levels.
  • Fig. 35 provides alterations in serum glycomic patterns between matched healthy (A) and cancer (B) patients
  • Fig. 36 provides a schematic representation of an example of the computational strategy for the analysis of glycoprofile patterns.
  • carbohydrates play a signficant role in a variety of biological and pathological processes. However, information regarding which carbohydrates are important and how they affect biological functions is limited. Therefore, additional methods for analyzing carbohydrates are desirable. Some of the methods provided herein provide better limits of detection of glycans and/or glycoconjugates that, in some examples, can extend to lower than 5fmol. Methods are provided herein which are directed to improved methods of analyzing carbohydrates.
  • the term "carbohydrate” is intended to include any of a class of aldehyde or ketone derivatives of polyhydric alcohols. Therefore, carbohydrates include starches, celluloses, gums and saccharides.
  • saccharides include mono-, di-, tri- and polysaccharides (or glycan).
  • Glycans can be branched or branched. Glycans can be found covalently linked to non-saccharide moieties, such as lipids or proteins (as a glycoconjugate).
  • covalent conjugates include glycoproteins, glycopeptides, peptidoglycans, proteoglycans, glycolipids and lipopolysaccharides.
  • glycoproteins glycopeptides
  • peptidoglycans proteoglycans
  • glycolipids glycolipids and lipopolysaccharides.
  • the use of any one of these terms also is not intended to be limiting as the description is provided for illustrative purposes.
  • the glycans can also be in free form (i.e., separate from and not associated with another moiety).
  • the use of the term peptide is not intended to be limiting.
  • the method provided herein are also intended to include proteins where "peptide" is recited.
  • the methods can be used to analyze glycans that are found as part of a glyconjugate or are found in free form.
  • the methods provided are also directed to the analysis of the total glycome of a sample.
  • the sample can be of a cell, group of cells, tissue or serum.
  • the "total glycome” refers to all of the glycans that can found in a sample.
  • the glycans can be in free form or they can be part of one or more glycoconjugates in the sample.
  • the total glycome therefore, represents all of the glycans (in free form, as part of glycoconjugates or both) in the sample.
  • a sample of glycans or the like is intended to include a sample containing free glycans and/or glycans as part of glycoconjugates.
  • the sample can be, for instance, a sample of body fluid. Samples of body fluid include serum, plasma, blood, urine, saliva, sputum, tears, CSF, seminal fluid, feces, etc.
  • the sample can also be, as an example, a sample of a cell, group of cells or tissue.
  • Glycans include N- and O-glycans.
  • N-glycans are classified into three types based on their structure: high mannose, hybrid and complex [ Sears, P., Wong, CH. (1998) Enzyme action in glycoprotein synthesis. Cell Mol Life Sci 54, 223-52.] All N-glycans contain a conserved pentasaccharide core composed of two N-acetylglucosamine residues followed by three mannose saccharides (Fig. 1). High mannose structures contain up to six more mannoses on both branches (Fig. 2A), while complex structures have no additional mannoses on either arm (Fig.2B). Instead, they are composed of additional hexosamines and/or galactose.
  • Hybrid structures are mixes of both high mannose and complex structures (Fig. 2C). Additionally, branch termini can be capped with sialic acid (a charged monosaccharide), and the core or branches can be fucosylated. Many other rare modifications exist, including sulfate, phosphate and xylose, but these are typically not found in humans.
  • the methods of analyzing glycans may include the analysis of any glycan including glycans with any of the structures described herein.
  • the term "glycan" is also intended to include glycans that are intact (i.e., as they were originally found in a sample) or have been digested (i.e., fragment of the original glycan).
  • Glycans can be analyzed with a number of different methods that include different steps and different experimental techniques.
  • the glycans can be, for example, those displayed on proteins or lipids, on the surface of cells or any of the glycans that are present in a body fluid.
  • the sample of glycoconjugates can be first denatured with a denaturing agent.
  • a "denaturing agent” is an agent that alters the structure of a molecule, such as a protein. Denaturing agents, therefore, include agents that cause a molecule, such as a protein to unfold. Denaturing can be accomplished with any of a number of methods that are known in the art.
  • Denaturing can be accomplished, for instance, with heat, with heat denaturation in the presence of ⁇ -mercaptoethanol and/or SDS, by reduction followed by carboxymethylation (or alkylation), etc.
  • Reduction can be accomplished with reducing agent, such as, dithiothreitol (DTT).
  • DTT dithiothreitol
  • Carboxymethylation or alkylation can be accomplished with, for example, iodoacetic acid or iodoacetamide.
  • Denaturation can, for example, be accomplished by reducing with DTT, ⁇ -mercaptoethanol or tri(2-carboxyethyl)phosphine (TCEP) followed by carboxymethylation with iodoacetic acid.
  • the denaturation can be accomplished with EndoF.
  • the glycoconjugates can also be denatured with denaturing agents, such as detergent, urea or guanidium hydrochloride.
  • denaturing agents such as detergent, urea or guanidium hydrochloride.
  • the sample of glycoconjugates is reduced with a reducing agent.
  • reducing agents include DTT, ⁇ -mercaptoethanol and tri(2-carboxyethyl)phosphine (TCEP).
  • TCEP tri(2-carboxyethyl)phosphine
  • the sample of glycoconjugates is alkylated after being reduced, such as, for example, with iodoacetic acid or iodoacetamide.
  • Methods of analyzing glycans of glycoconjugates can also include cleaving the glycans from the non-saccharide moiety using any chemical or enzymatic methods or combinations thereof that are known in the art.
  • An example of a chemical method for cleaving glycans from glycoconjugates is hydrazinolysis or alkali borohydrate.
  • Enyzmatic methods include methods that are specific to N- or O-linked sugars. These enzymatic methods include the use of Endoglycosidase H (Endo H), Endoglycosidase F (EndoF), N- Glycanase F (PNGaseF) or combinations thereof.
  • PNGaseF is used when the release of N-glycans is desired.
  • PNGaseF is used for glycan release the proteins is, for example, first unfolded prior to the use of the enzyme.
  • the unfolding of the protein can be accomplished with any of the denaturing agents provided above.
  • the glycans analyzed by the methods provided herein can also be contacted with a glycan-degrading enzyme.
  • glycan-degrading enzymes are known in the art and include sialidase, galactosidase, mannosidase, N-acetylglucosaminidase or a combination thereof.
  • the methods provided herein also include the use of a carbohydrate-degrading enzyme.
  • carbohydrate-degrading enzymes or "glycan-degrading enzymes” are enzymes that can modify a carbohydrate or glycan in some way.
  • Some examples of glycan-degrading enzymes include sialidase, galactosidase, mannosidase, N- acetylglucosaminidase or some combination thereof.
  • samples can also be purified with commercially available resins and cartridges for clean-up after chemical cleavage or enzymatic digestion used to separate glycans from protein.
  • resins and cartridges include ion exchange resins and purification columns, such as GlycoClean H, S, and R cartridges.
  • GlycoClean H is used for purification.
  • Purification can also include the removal of high abundance proteins, such as the removal of albumin and/or antibodies, from a sample containing glycans.
  • the purification can also include the removal of unglycosylated molecules, such as unglycosylated proteins. Removal of high abundance proteins can be a desirable step for some methods, such as some high-throughput methods described elsewhere herein.
  • abundant proteins, such as albumin or antibodies can be removed from the samples prior to the final composition analysis.
  • the sample of glycans Prior to the analysis of a sample of glycans as provided herein the sample of glycans can also be fractionated. The sample can be fractionated so as to obtain a sample of glycans with specific subgroups of molecules.
  • Subgroups of molecules include molecules of specific properties, such as charge ⁇ molecular weight, size, binding properties to other molecules or materials, acidity, basicity, pi, hydrophobicity, hydrophilicity, etc.
  • the subgroup of molecules of a sample is the low abundance species, and it is the low abundance species that are analyzed with the methods.
  • the low abundance species can contain fucoses, sialic acids, galactoses, mannoses or sulfate groups.
  • the fractionation can be performed using any methods known in the art. Such methods include using solid supports with immobilized proteins, organic molecules, inorganic molecules, lipids, carbohydrates, nucleic acids, etc.
  • the fractionation can also be performed with filters, such as molecular weight cutoff filters, resions, such as cation or anion exchange resins, etc. Therefore, the method provided herein can be used for the analysis of the glycans of a subgroup of molecules.
  • Glycans can be charged or uncharged. They can be acidic, basic or neutral. It has now been found that separately analyzing charged and uncharged glycans of a sample can provide an improvement in the analysis of glycans. Therefore, the charged and uncharged glycans can be separated prior to the analysis of the glycans, such as with an analytic method.
  • any of the methods provided herein can include a step of separating neutral and charged glycans, such as acidic glycans.
  • Such separation can be achieved using purification methods. For instance, in a preferred embodiment, the separation is accomplished with a porous carbon purification cartridge by eluting glycan pools with different concentrations of acetonitrile. Other methods will be known to those of skill in the art. Analysis of these separate glycan pools can then be undertaken.
  • the acidic glycans can be analyzed in negative ion mode, while the neutral glycans are analyzed in positive ion mode.
  • the glycans can be modified to improve ionization of the glycans, particularly when MALDI-MS is used for analysis. Such modifications include permethylation.
  • An other method to increase glycan ionization is to conjugate the glycan to a peptide. Examples of the methods are described further in the Examples below.
  • spot methods can be employed to improve signal intensity.
  • any analytic method for analyzing the glycans so as to characterize them can be performed on any sample of glycans, such analytic methods include those described herein.
  • to "characterize" a glycan or other molecule means to obtain data that can be used to determine its identity, structure, composition or quantity.
  • the term can also include determining the glycosylation sites the glycosylation site occupancy, the identity, structure, composition or quantity of the glycan and/or non-saccharide moiety of the glycoconjugate as well as the identity and quantity of the specific glycoform.
  • These methods include, for example, mass spectrometry, NMR (e.g., 2D-NMR), electrophoresis and chromatographic methods.
  • mass spectrometic methods include FAB-MS, LC-MS, LC-MS/-MS, MALDI-MS, MALDI-TOF, TANDEM- MS, FTMS, etc.
  • NMR methods can include, for example, COSY, TOCSY, NOESY.
  • Electrophoresis can include, for example, CE-LIF.
  • the glycans can be quantified using calibration curves of known glycan standards. More details regarding examples of these methods are provide below in the Examples.
  • FACE fluorescence assisted carbohydrate electrophoresis
  • CE capillary electrophoresis
  • a method for the compositional analysis of oligosaccharides using CE has been described (Rhomberg, A. J., Ernst, S., Sasisekharan, R. & Biemann, K. (1998) Proc Natl Acad Sci USA 95, 4176-81).
  • the analytic method for the characterization of the glycans includes the use of MALDI-MS.
  • Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) techniques for the analysis of oligosaccharides have also been described (Juhasz, P. & Biemann, K. (1995) Carbohydr Res 270, 131-47 and Juhasz, P. & Biemann, K. (1994) Proc Natl Acad Sci USA 91, 4333-7; Venkataraman, G., Shriver, Z., Raman, R. & Sasisekharan, R. (1999) Science 286, 537-42; Rhomberg, A. J., Shriver, Z., Biemann, K. & Sasisekharan, R. (1998) Proc Natl Acad Sci USA 95, 12232-7; Ernst, S.,
  • Analytic methods can also comprise the use of carbohydrate- or glycan-degrading enzymes. Following enzymatic degradation the sample of degraded glycans can be further analyzed with an analytic method as described above or otherwise known in the art.
  • the characterization of one or more glycoconjugates with an analytic method can also include determining the identity, structure or sequence of the non-saccharide moiety of the glycoconjugate.
  • the characterization with an analytic method can include determining the peptide (or lipid) sequence of a glycopeptide (or glycolipid).
  • the matrix in which the sample of glycans is suspended may affect the quality of compositional analysis.
  • the matrix preparation is caffeic acid with or without spermine.
  • the matrix preparation is DHB with or without spermine.
  • the matrix preparation is spermine with DHB.
  • the spermine for example, can be in the matrix preparation at a concentration of 300 mM.
  • the matrix preparation can also be a combination of DHB, spermine and acetonitrile.
  • MALDI-MS can also be performed in the presence of Nafion and ATT. Additionally, when using MALDI-MS to analyze the samples, instrument parameters can also be modified. These parameters may include guide wire voltage, accelerating voltage, grid values and negative versus positive mode.
  • the samples of glycans can be analyzed separately or they can be analyzed as a mixture.
  • the methods provided include methods for the analysis of glycosylation of a single protein (or lipid) in a sample or a mixture of proteins (or lipids or a mixture of proteins and lipids).
  • glycoconjugate such as a glycoprotein
  • a glycoconjugate can exist in many glycoforms; that is, each glycosylation site may (or may not) be occupied by a specific glycan all the time.
  • the methods provided comprise or consist of steps for determining the glycosylation site occupancy of the glycoconjugates of a sample.
  • glycosylation site occupancy refers to the frequency (percentage) in which one or more specific glycosylation sites on a lipid, protein or peptide is occupied by a glycan.
  • the glycosylation site can be determined using the methods provided below in the Examples as well as methods that are known in the art.
  • the glycosylation site occupancy is the "total glycosylation site occupancy", which refers to the frequencies in which all of the specific glycosylation sites on a lipid, protein or peptide are occupied by a glycan.
  • the specific glycans that occupy each specific glycosylation site can also be characterized using one or more analytic techniques.
  • 2D-NMR provides a reliable method for the identification of N-linked and O-linked glycan site occupancy.
  • a combination of COSY, TOCS Y, NOES Y experiments are first conducted on a specific quantity of a glycoprotein.
  • Fig. 24 provides one embodiment of a method for determining glycosylation site occupancy. Briefly, a well characterized batch of the glycoprotein under study is used to generate a library of labeled peptides and glycopeptides by trypsin digest. In order to order to facilitate the determination of the glycosylation sites, each glycosylated amino acid is differentially labeled.
  • the labels that can be used include isotopes of C, N, H, S, or O.
  • the glycosylated amino acids are labeled with O 18 and O 16 using methods known in the art. [Kaji, 2003].
  • the samples can be further analyzed.
  • the glycan site occupancy is quantified from the ratios of the masses of the labeled and unlabeled fragments.
  • determining the glycosylation site and its occupancy can include cleaving and labeling with a first label the glycoconjugates at the glycosylation sites of a portion of the sample, cleaving the glycoconjugates at the glycosylation sites of another portion of the sample and analyzing the portion of the sample.
  • the portions of the sample can be analyzed separately or as a mixture in any ratio. First instance, when there are two portions of the sample, the two portions can be mixed in a 1:1, 1 :2, 1 :3, 1 :4, or 1 :5 ratio.
  • the glycosylation site occupancy method can be used to determine the identity and number of glycoforms in the sample. Therefore, a method of determining the identity and number of glycoforms in a sample comprising determining the glycosylation site occupancy of a glycoconjugate and analysis to characterize the glycoconjugates so as to determine the identity and number of glycoforms is also provided. As illustrated in the Examples below, the fragment containing the partner peak with a molecular weight 2Da heavier is identified as the peptide containing the glycosylation site.
  • a preliminary identification all the peptides (or lipids when the glycoconjugate is a glycolipid) and glycopeptides (or glycolipid) are identified and a preliminary identification of the glycans is obtained.
  • This quantitative information can be combined with a glycan analysis and used as constraints in a computational analysis, such as the one described below, txrarrive at the complete characterization of the glycoprotein.
  • Constraints as used herein are one or more values or relationships to which results obtained from an analysis of a sample containing glycans can be compared to or evaluated.
  • the constraints can, for example, be one or more mathematical equations that can be solved with the data obtained from an analysis of a sample containing glycans and/or other data obtained from other sources, such as databases or with other analytical tools.
  • the constraints can, for example, be generated from the one or more of the results obtained from an analysis of a sample of glycans and/or with other glycan/glycoconjugate information, such as the information regarding glycan synthesis or from databases.
  • the labels that can be used in any of the methods provided include isotopes of C, N, H, S, or O. In one embodiment the glycosylated amino acids are labeled with O . Also provided is a method of generating a library.
  • the library consists of labeled glycoconjugates and fragments of the glycoconjugates, the fragments being the non- saccharide portions of the glycoconjugates.
  • a library is generated by cleaving the backbone of the glycoconjugate and labeling the non-saccharide fragments and and the non-saccharide portions of the glycoconjugates that result with a labeling agent.
  • This example also includes the step of cleaving the glycans from the glycoconjugate. The glycans can then be removed from the sample.
  • the libraries so produced can be analyzed with the methods provided herein.
  • the libraries can also be used as a standard once characterized and methods of using such libraries are also provided.
  • a method of analyzing a sample with glycoconjugates includes cleaving the glycoconjugates, enzymatically removing the glycans from the glycoconjugates and mixing the sample with a standard. The sample mixed with the standard can then be analyzed. In one embodiment, the amounts of the glycoconjugates and non-saccharide moieties of the sample and standard are compared. In one aspect of the invention the standards are also provided.
  • the methods provided can also comprise or consist of the steps of generating a list of glycan properties.
  • One example of such a method includes measuring 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more properties of the glycan, recording a value for the one or more properties to generate a list of the glycan properties.
  • the method also is intended to refer to generating a list of glycoconjugate properties.
  • a "property” as used herein is a characteristic (e.g., structural characteristic) of the glycan or glycoconjugate that provides information (e.g., structural information) about the glycan or glycoconjugate. Examples of properties include ⁇ charge, chirality, nature of substituents, quantity of substituents, molecular weight, molecular length, compositional ratios of substituents or units, type of basic building blocks (saccharide, amino acid, lipid constituents), hydrophobicity, enzymatic sensitivity, hydrophilicity, secondary structure and conformation, ratio of one set of modifications to another set of modifications, etc.
  • the list comprises the number of one or more types of monosaccharides.
  • the list can also include the total mass of the glycan or glycoconjugate, the mass of the non-saccharide moiety of a glycoconjugate, the mass of one and/or more modified glycans, etc.
  • the list in one embodiment can be a data structure tangibly embodied in a computer-readable medium, such as computer hard drive, floppy disk, CD-ROM, etc.
  • Table 6 represents an examples for such a data structure.
  • the data structure of Table 6 has a plurality of entries, where each entry encodes a value of a property.
  • the values encoded can be encoded by any kind of value, for example, as single-bit values, single-digit hexadecimal values, or decimal values. Therefore, also provided is a database, tangibly embodied in a computer-readable medium, wherein the database stores information descriptive of one or more glycans and/or glycoconjugates.
  • the database comprises data units that correspond to the glycan and/or glycoconjugate.
  • the data units include an identifier that includes one or more fields, each field storing a value corresponding to one or more properties of the glycans and/or glycoconjugates. In one embodiment the identifier includes 2, 3, 4, 5, 6, 7, 8, 9, 10 or more fields.
  • the database can be a database of all possible glycoconjugates, glycans or can be a database of values representing a glycome profile or pattern for one or more samples.
  • Methods of analyzing and characterizing a glycome profile or pattern is described further below.
  • improved methods for analyzing samples containing glycans are provided, which include a combined analytical-computational platform to achieve a thorough characterization of glycans. Therefore, any of the methods provided can be combined with computational methods.
  • Non-limiting examples of the computational methods that can be used are illustrated in detail in the Examples. Briefly, the diverse information gathered from the different experimental techniques can be used to generate constraints.
  • a method of analyzing glycans with the combined analytic and computational techniques can include the steps of performing an experiment on a sample containing glycans, analyzing the results of the experiment, generating constraints and solving them.
  • the constraints can be generated and/or solved with the data obtained from experimental results as well as other known information, such as information from databases that contain information about glycans or glycoconjugates and with other tools that analyze the properties of glycans, glycoconjugates or the non-saccharide moieties thereof, such as mass and enzyme action.
  • the constraints can be generated using, for instance, what is known of the biosythetic pathway of glycan synthesis. Unlike DNA or protein synthesis, which are template-driven processes, glycan biosynthesis is a complex process involving a multitude of enzymes. A detailed scheme of N-glycan biosynthesis is shown in Fig. 3 and the biosynthetic enzymes and their EC numbers are listed in Table 1.
  • the process is initiated in the cytoplasm, with the nascent sugar attached to the ER membrane through a lipid anchor. After a glycan core of two glucosamines followed by five mannose residues is constructed, the orientation of the growing glycan is flipped to face the lumen of the ER. There, four more mannose residues are added by ⁇ -mannosyltransferase, and one branch is capped with three glucoses. At this point, oligosaccharyl transferase catalyzes the removal of the naive glycan from its lipid anchor, and attaches it to a glycosylation site on a protein undergoing synthesis in the ER [ Varki, A.
  • Fig. 23 A more detailed illustration of one embodiment of an analytical and computational method is provided in Fig. 23.
  • experimental analytic methods can be combined with computational methods to achieve the desired characterization. It is the combination which provides more efficient analysis of samples of glycans.
  • the examples provided are not intended to be limiting in any way.
  • the methods provided herein also include generating a list of the possible compositions of glycans and their theoretical masses. The list can be based on the biosynthetic pathways for glycosylation (Fig. 3). An example of such a list is provided herein.
  • the list can also be recorded in a computer-readable medium.
  • the list can be generated with other means, such as with the results from the use of any of the methods provided herein or known in the art.
  • the method can include the use of exoenzymes to cleave the glycans in order to analyze the composition of the glycans.
  • the list can be used in any of the methods provided in order to characterize glycans. Methods that include the use of a list are also provided herein. Protein glycosylation can affect the function of proteins or be indicative of a cause or symptom of a disease state.
  • N- and O-linked glycans are an important factor for determining proper folding, stability and resistance to degradation (which affects the half-life of the protein).
  • N- and O-linked glycans play a role in the activity and/or function of the protein.
  • N- and O-linked glycans are indicative of a normal or disease state. Therefore, methods are provided herein to analyze the glycosylation of a protein for a variety of reasons. The methods, therefore, provided above can be used in diagnosis. Also provided is the method described below, which can also be used for diagnostic or prognostic purposes.
  • the total profile of carbohydrates from body fluids or tissues can be examined, and in some embodiments this can be done in a high-throughput format.
  • the examination of total glycan profiles are now exceedingly accessible thanks to the recent advances in proteomic pattern diagnostics.
  • This approach should be useful in sensing susceptible physiological alterations to the body's natural homeostasis.
  • This method should not only serve as a fast diagnostic tool but should also help to understand the function of specific carbohydrate modifications in some diseases.
  • proteins comprise an enormous portion of serum, approximately 7% of the total wet weight [Vander, 2001].
  • albumin ⁇ 50mg/ml
  • albumin a protein that can be non-enzymatically glycosylated, but not N-glycosylated
  • albumin can obscure analysis for proteomics, it may not interfere with N-glycan profiling.
  • glycosylated antibodies which have a number of glycan structures [Bihoreau, 1997; Watt, 2003].
  • simple methods exist to separate these abundant antibodies from the less abundant glycoproteins.
  • albumin does not have N-linked sugars
  • the sheer quantity present may interfere with glycan release or purification.
  • immunoglobulins There are several other major proteins in serum (i.e. immunoglobulins) that are N-glycosylated, which may overshadow the signals from less abundant proteins.
  • immunoglobulin glycosylation may also be correlated with changes in physiological state.
  • To determine the contributions and/or interference from major serum proteins several options for separating serum proteins into fractions before analysis were explored. Identifying glycan structures with complex protein mixtures can be somewhat difficult.
  • each mass peak corresponds uniquely to a monosaccharide assignment.
  • the method provided allows fast and sensitive spectrometric analysis of patterns for the total composition of glycans in body fluids such as serum, saliva, urine, tears, seminal fluid, feces, etc. Specifically the use of the analysis of these patterns can be extended for the purpose of diagnosis, prognosis and monitoring the effects of therapeutics.
  • body fluids such as serum, saliva, urine, tears, seminal fluid, feces, etc.
  • the use of the analysis of these patterns can be extended for the purpose of diagnosis, prognosis and monitoring the effects of therapeutics.
  • the total content of serum, saliva and urine glycome was analyzed and it was shown that specific and reproducible MALDI-MS patterns which are dependent on the source (patient) of the sample and state could be obtained. Since every signal inside the pattern corresponds to specific glycans, the alteration of these patterns are easily determined and correlated with the expression levels of the carbohydrates. These alterations can be easily determined manually or more efficiently with the help of computational analysis.
  • this method serve as reliable platform for diagnosis, prognosis and the analysis associated with therapeutics.
  • the methods provided can also be used to profile populations to aid the development and application of patient-oriented treatments. Methods, therefore, are provided for the determining the glycome profile of a sample.
  • the total glycome and/or patterns deduced thefefrom can be used for studying the effects of glycosylation on protein activity and/or function as in the case of glycoprotein therapeutics.
  • the total glycome and/or patterns deduced therefrom can also be used in methods for diagnosis, assessing prognosis and assessing drug treatment, etc.
  • a "glycome profile" refers to the number and kind of glycans found in a sample.
  • the sample can contain one or more glycans and/or one or more glycoconjugates.
  • the glycome profile can be, for example, the number and kind of a specific type of glycan (e.g., N-glycan, O-glycan, etc.). Each component of a glycome profile can correspond to a glycan or fragment thereof or a glycoconjugate or fragment thereof. The number refers to the amount and can be an actual or a relative amount.
  • the "total glycome profile" as used herein the absolute or relative number and kind of all glycans in a sample.
  • the sample can be a sample of cells, tissue or body fluid. To assess the glycome profile of a sample any analytic methods can be used.
  • the analytic method can be MALDI-MS, LC-MS, LC-MS/MS, MALDI-TOF, TANDEM-MS, FTMS, NMR, HPLC, electrophoresis, capillary electrophoresis, microfluidic devices or nanofluidic devices.
  • the glycome profile is determined using a quantitative MALDI-MS or MALDI-FTMS in the presence of ATT and Nafion coating.
  • calibration curves of known glycan standards can be used. Prior to analyzing the glycans of the sample, the sample can be fractionated.
  • the sample can be fractionated based on properties of the glycans and/or glycoconjugates, such as but not limited to, charge, size, molecular weight, binding properties to other molecules or materials, acidity, basicity, pi, hydrophobicity and hydrophilicity.
  • the fractionation can be performed using solid supports with immobilized proteins, organic molecules, inorganic molecules, lipids, carbohydrates, nucleic acids, etc.
  • the fractionation can be performed using filters, such as molecular weight cutoff filters.
  • the fractionation can also be performed using resins, such as, cation or anion exchange resins. Any method of fractionation known in the art can be used. In one embodiment, however, the sample is not fractionated before it is analyzed.
  • the sample Prior to analysis the sample, the sample can also be degraded with a chemical or enzymatic method to cleave the glycans from any glycoconjugates in the sample.
  • enzymatic methods are provided above and include, for example, the use of PNGase F, endoglycosydase H and endoglycosydase F or combinations thereof.
  • Chemical methods have also been described above and include hydrazinolisis or alkali borohydrate. After chemical or enzymatic degradation the sample can then be performed in some embodiments. Purification methods were also provided above. Examples of particular purification methods include using solid phase extraction cartridges, such as graphitized carbon columns and C-18 columns. Once a glycome profile is determined, a glycome pattern can be identified.
  • glycome pattern refers to a glycome profile or subset of the profile that has been associated with a certain function (of a lipid or protein), cellular state, or pathological condition (i.e., a disease condition).
  • a glycome pattern can be identified using a computational method.
  • An glycome pattern, like, the profile can be represented by the relative or absolute amounts of components of the pattern or ratios between the components of the pattern.
  • the glycome pattern can also be represented by combinations of different components or the presence or absence of a component.
  • the pattern can also be any combination of respresentations, such as those provided herein.
  • the pattern can be determined using a computational method. Examples of such computational methods are provided herein in the Examples.
  • the computational method can, for example, incorporate one or more of the following to determine a glycome pattern: experimental data from analytic methods of glycome and/or glycan analysis; theoretical glycan structures; glycan composition, structure, property information from databases, glycan biosynthetic pathway information, patient or sample origin information, such as patient history, demographics; extracting features from the experimental data sets, generating all possible data sets with specific features, submitting the combined information to a data mining analysis, establish the relationship rules and validating the pattern.
  • the computational method can be an iterative process.
  • One detailed example is provided in Fig. 3.
  • the patterns that are ultimately validated can be recorded in a computer-generated data structure.
  • a database of validated glycome patterns is, therefore, also provided herein.
  • the patterns can be subsequently used for, for example, diagnostic and prognostic purposes and for determining the purity of a sample.
  • the methods provided herein include methods for determining the glycosylation of a protein and its effects on the protein's activity and/or function.
  • the protein glycosylation can be studied with the methods provided to determine the proper folding of the protein or to determined the influence of the protein's glycosylation on the stability/and or degradation resistance of the protein (indicative of the protein's half-life). Changing the composition or the degree of glycosylation of a protein can greatly influence its half-life in circulation, as well as its activity [Chang, G.D., Chen, C.J., Lin, C.Y., Chen, H.C, Chen, H.
  • EPO erythropoietin
  • glycosylation plays an important role in the structure of the Fc region, which is important for activation of leukocytes expressing Fc receptors.
  • IgG glycosylation is species specific, making it essential to choose the appropriate production method for protein therapeutics [Raju, T.S., Briggs, J.B., Borge, S.M., Jones, A.J.
  • glycoprotein therapeutics a human protein produced in a mouse cell line may not have the necessary glycans for optimal function in human patients. Therefore, the immune recognition of an antibody can be assessed with the methods of analysis provided herein.
  • One of the major challenges during the production of glycoprotein therapeutics is to control the generation of a specific glycoform and the subsequent characterization for quality control of the product. Therefore, methods that can efficiently characterize new batches of glycoprotein therapeutics are of great value to the pharmaceutical industry.
  • glycoprotein therapeutics For a complete characterization of glycoprotein therapeutics, information such as glycan site occupancy, carbohydrate composition and structure at each site and quantity of each carbohydrate is required. As described below in the Examples, the methods for analyzing glycans found on proteins, which can include antibodies, can be used to assess the quality and variability of protein production. With the recently increased focus on protein-based therapeutics by pharmaceutical companies and research laboratories, it has become important to understand how glycosylation composition is influenced by protein production methods. In the field of bioprocess engineering, there are many different types of bioreactors available for protein production. Depending on the model, parameters such as pH and dissolved oxygen (DO) can be controlled in several ways, and agitation methods can result in wide variations in shear stress.
  • pH and dissolved oxygen (DO) can be controlled in several ways, and agitation methods can result in wide variations in shear stress.
  • the cell-feeding process during fermentation can be altered to change the cell growth profile. All of these variables can affect protein glycosylation — even using identical conditions in two different bioreactors causes changes in glycan patterns [Kunkel, J.P., Jan, D.C, Butler, M., Jamieson, J.C (2000) Comparisons of the glycosylation of a monoclonal antibody produced under nominally identical cell culture conditions in two different bioreactors. Biotechnol Prog 16, 462-70; Zhang, F., Saarinen, M.A., Itle, L.J., Lang, S.C, Murhammer, D.W., Linhardt, R.J.
  • a well characterized batch of the glycoprotein under study is used to generate a library of backbone-labeled peptides and glycopeptides by enzymatic digest using methods know in the art [Gehrmann, 2004;Yao, 2003;Reynolds, 2002; Yao, 2001]. Trypsin proteolytic digest cleavage can be employed before and after glycan cleavage in order to expand the peptide library. Peptide labeling can be performed using methods know to experts in the art. Each characterized and quantified peptide and glycopeptide can be used to generate calibration curves using LC-MS or LC- MS/MS techniques.
  • peptides and glycopeptides can then be mixed (in known concentrations) with the petide/glycopeptide mixture resulting from the trypsin proteolytic cleavage digest of the new sample batch under study.
  • the co-elution of the labeled peptides with the unknown peptides followed by the co-detection (the ratio between labeled and unlabeled peptides) using mass spectrometry allows the quantification of each peptide (and therefore the different glycoforms) in the unknown sample.
  • the respective glycans from the eluted glycopeptides can be analyzed using the methods described herein.
  • the use of other well established methods e.g., hydrazide column, peptide
  • the methods provided, where the amount or type of glycans on proteins can be determined, can be used to analyze the purity of a protein sample.
  • the term "purity" refers to the proportion of a protein sample that contains a particular glycan or a particular glycosylation pattern.
  • the protein sample is determined to be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more pure.
  • the method is used to assess the amount of a particular glycan in a protein sample.
  • it may be desired that the proteins are selected depending on the particular glycosylation pattern they exhibit.
  • “glycosylation” is meant to include the pattern or a subset or even one particular glycan, while “glycosylation pattern” refers to the number and kind of glycans present on the protein.
  • the methods provided herein can be used to evaluate a process of producing proteins and/or compare a process with another to evaluate the types of proteins produced.
  • the "types of proteins produced" includes not only the protein itself but also its glycosylation pattern. As stated above, the glycosylation of a protein may be indicative of a normal or a disease state. Therefore, methods are provided for diagnostic purposes based on the analysis of the glycosylation of a protein or set of proteins, such as the total glycome.
  • the methods provided herein can be used for the diagnosis of any disease or condition that is caused or results in changes in protein glycosylation. For example, the methods provided can be used in the diagnosis of cancer, inflammatory disease, benign prostatic hyperplasia (BPH), etc.
  • the diagnosis can be carried out in a person with or thought to have a disease or condition.
  • the diagnosis can also be carried out in a person thought to be at risk for a disease or condition.
  • "A person at risk” is one that has either a genetic predisposition to have the disease or condition or is one that has been exposed to a factor that could increase his/her risk of developing the disease or condition.
  • the person can have, be thought to have or is at risk of cancer, cystic fibrosis, mad cow disease, etc. Detection of cancers at an early stage is crucial for its efficient treatment. Despite advances in diagnostic technologies, many cases of cancer are not diagnosed and treated until the malignant cells have invaded the surrounding tissue or metastasized throughout the body.
  • Cancers or tumors also include but are not limited to adrenal gland cancer, biliary tract cancer; bladder cancer, brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; extrahepatic bile duct cancer; gastric cancer; head and neck cancer; intraepithelial neoplasms; kidney cancer; leukemia; lymphomas; liver cancer; lung cancer (e.g.
  • Protein samples may include samples from a subject.
  • the samples can, for example, be serum or saliva samples.
  • the methods can also be used to determine whether or not cells are undergoing dramatic change or are "stressed cells". Stressed cells are cells that are undergoing a stress response that alters the cell's protein production. The stress response can be any change that causes altered protein production or causes the cell to deviate from its normal state. Stressed cells can be identified by analyzing the glycans exhibited by the proteins on the cell's surface.
  • Such glycans can be found in, for example, a glycoprotein.
  • the methods provided are used to detect changes in glycosylation that occur under growth conditions or inflammation.
  • methods for analyzing blood type antigens are also provided.
  • methods for therapeutics are provided for therapeutics.
  • the glycosylation of proteins can be assessed to evaluate treatment regimens and/or to select specific therapies.
  • a subject is any human or non-human vertebrate, e.g., dog, cat, horse, cow, pig.
  • a sample includes any sample obtained from any of these subjects.
  • High-throughput methods are also provided. "High-throughput" methods refer to the ability to process and/or analyze multiple samples at one time.
  • High-throughput methods provided herein can include the use of a membrane-based method, such as a PVDF membrane in a 96-well plate, for high throughput sample processing (i.e., digestion and/or denaturation steps, etc.)
  • membrane based high-throughput methods may also include the removal of abundant proteins such as albumin.
  • the methods of analysis provided are high-throughput methods. Any step or steps of any of the methods provided herein can be performed as a high- throughput step. For instance purification, degradation, etc. can be performed in a high- throughput manner in some embodiments.
  • Robotics can be used in one or more steps of the methods provided herein. In one embodiment robotics is used for separation.
  • PNGaseF digest of N-Glycans from Protein Cores Between 10 and lOO ⁇ g of protein was denatured for 10 minutes at 90°C with 0.5% SDS and 1% ⁇ -mercaptoethanol. Since SDS (and other ionic detergents) inhibits enzyme activity, 1% NP-40 was added to counteract these effects. The enzyme reaction was performed overnight with 2 ⁇ l of PNGaseF at 37°C in a 50mM sodium phosphate buffer, pH
  • GlycoClean R and S cartridges were purchased from Prozyme (San Leandro, CA; formerly Glyko). GlycoClean R cartridges were primed with 3ml of 5% acetic acid, and the samples were loaded in water. Sugars were eluted with 3ml of water passed through the column. For GlycoClean S, the membrane was primed with 1ml water and 1ml 30% acetic acid, followed by 1ml acetonitrile. The glycan sample was loaded (in a maximum volume of lO ⁇ l) onto the disc, and the glycans were allowed to adsorb for 15 minutes.
  • GlycoClean H cartridges were purchased from Prozyme (200mg bed) or ThermoHypersil (25mg bed). To prepare the GlycoClean H cartridge, the column (containing 200mg of matrix) was washed with 3ml of IM NaOH, 3ml H 2 O, 3ml 30% acetic acid, and 3ml H 2 O to remove impurities.
  • the matrix was primed with 3ml 50% acetonitrile with 0.1% TFA (Solvent A) followed by 3ml 5% acetonitrile with 0.1% TFA (Solvent B). After loading the sample in water, the column was washed with 3ml H 2 O and 3ml Solvent B. Finally, the sugars were eluted using 4x0.5ml of Solvent A.
  • GlycoClean H cartridges can be reused after washing with 100% acetonitrile and re-priming with 3ml of Solvent A followed by 3ml of Solvent B. For the 25mg cartridge, wash volumes were reduced to 0.5ml. Eluted fractions were lyophilized and the isolated glycans were resuspended in 10-40 ⁇ l H 2 O.
  • MALDI-MS of N-Glycans Several MALDI-MS matrix compounds were tested in this study.
  • caffeic acid was added to 30% acetonitrile to make a saturated solution, with or without 300 mM spermine.
  • a saturated solution of dihydroxybenzoic acid (DHB) in water was used with or without 300 mM spermine.
  • DRB dihydroxybenzoic acid
  • Endoglycosidases H and F (EndoH and EndoF) cleave between the two interior Glc ⁇ Ac residues of the glycan core, while protein N-glycanase F (PNGaseF) cleaves between the interior GlcNAc and the asparagine side chain of the protein core [32-34].
  • EndoH only acts on high mannose or hybrid structures, while EndoF can cleave complex glycans. With EndoH and EndoF, information about fucosylation on the reducing end GlcNAc is lost since this residue remains attached to the protein core.
  • PNGaseF releases the entire glycans and can cleave all classes of N-glycans, making it a tool of choice for N-glycan release.
  • proteins should be unfolded prior to digestion with PNGaseF.
  • a protein sample can be denatured by heating in the presence of ⁇ - mercaptoethanol and/or SDS.
  • samples contain a mixture of free glycans, protein, detergent (from the denaturing step), and salts. In some instance it is preferred that everything except the glycans are removed from the sample.
  • the proteins were first precipitated with ethanol and the supernatant containing the glycans was then dried under vacuum (SpeedVac) and resuspended in water. At this point, the most difficult component to get rid of was the detergent, which interferes with some types of analytical techniques.
  • Glyco R contains a reverse phase material that allows glycans to flow through, while retaining peptides and detergents.
  • Glyco S is a small membrane that adsorbs the sugars in >90% acetonitrile, while hydrophobic molecules are washed away. The glycans can then be eluted with water.
  • Glyco H is a porous graphitic carbon matrix which retains both neutral and charged sugars, while allowing salts to be washed away with a low concentration of acetonitrile. The sugars can then be eluted with higher acetonitrile concentrations. Proteins and detergents typically remain on the Glyco H column. Overall, the Glyco H cartridge yielded the best results in these studies (Table 2).
  • ⁇ MR can provide detailed structural information in a single experiment. Due to the lack of natural chromophores in N-linked carbohydrates, many of the procedures require the labeling of saccharides with chemical tags or fluorescent labels to facilitate detection. In fluorescence assisted carbohydrate electrophoresis (FACE), glycans are fluorescently labeled and run on a polyacrylamide gel [35]. Glycan bands can then be excised for further structural analysis. Similar methods use HPLC or capillary electrophoresis (CE) for greater sensitivity and better separation.
  • FACE fluorescence assisted carbohydrate electrophoresis
  • MALDI-MS is a soft ionization technique that utilizes an organic matrix to absorb and transfer the ionizing energy from the laser. This technique is useful for many applications, from small molecules to large proteins over 100 kDa.
  • sample ionization is sensitive to instrument conditions as well as sample preparation. In particular, the matrix used to suspend the sample is important for good ionization. The efficiency of a particular matrix can vary widely, depending on the nature of the sample.
  • FIG. 5 shows spectra of some of the representative RNaseB samples from Table 2, with glycans purified under different conditions. In the cleanest samples, all glycan masses correspond with high mannose structures (Man-5 through Man-9).
  • ovalbumin was used as a protein standard with complex type N-glycans. Optimized purification and MALDI-MS conditions were used (Glyco H 200mg, DHB matrix with spermine). The MALDI-MS data displayed results comparable to previously published reports [30].
  • Fig. 6 shows the MALDI-MS spectrum of ovalbumin glycans, and Table 3 lists the observed peaks and their structures.
  • pH can be controlled automatically by the instrument, which dispenses CO 2 , NaHCO 3 and O 2 as needed.
  • measurements must be taken manually and pH adjusted by hand.
  • the pH in this reactor can be controlled by either adding fresh media as the cells grow, or adding NaHCO 3 for increased buffering capacity, and CO 2 as needed.
  • the main difference between the reactor types is the mode of agitation.
  • a blade stirrer keeps the cell suspension in motion, while a sparger introduces oxygen to the system in a controlled manner.
  • a rocking motion generates waves that mix the components of the system and aids the transfer of oxygen and other gases into the system.
  • the purified antibodies were processed according to the optimized method described above.
  • lOO ⁇ g of protein was used as the starting material. Both positive and negative ion modes were used in the MALDI-MS to determine whether there were charged sugars present. No signal was observed in the negative mode, indicating that only neutral sugars were obtained from the antibodies.
  • the positive ion mode MALDI-MS data of the five antibody samples are shown in Fig. 7.
  • Glycoproteins produced using different conditions are shown in Table 4. All fractions contained the same six glycans at 1317 Da, 1463 Da, 1478 Da, 1625 Da, 1641 Da and 1787 Da. The structures corresponding to these peaks are shown in Fig.8 with their theoretical masses.
  • CD22 a B cell- specific immunoglobulin superfamily member, is a sialic acid-binding lectin. JBiol Chem 268, 7011-8.
  • N-Glycans After glycans were cleaved from the protein, the sample was dialyzed against water to remove excess salts and glycerol (from PNGaseF formulation). Samples were then spun for 5 minutes at 6000xg to remove most proteins and the supernatant lyophilized to ⁇ 500 ⁇ l. A C18 cartridge (Waters Corporation, Milford, MA) was primed with 3ml methanol, then 3ml water, and 3ml 5% acetonitrile with 0.1% TFA.
  • ConA buffer (20mM Tris, ImM MgCl 2 , lmM CaCl 2 , 500mMNaCl, pH 7.4).
  • 500 ⁇ l of serum was mixed with 150 ⁇ l of 5X ConA buffer.
  • glycoproteins were eluted with 2ml of 500mM ⁇ -methyl-mannoside and dialyzed against lOmM phosphate pH 7.2 overnight at 4°C
  • Protein A-agarose beads were purchased from Calbiochem (La Jolla, CA). Before use, 1ml beads were washed 3X with PBS. To separate IgG from other serum proteins, samples were diluted 1 :4 with PBS and incubated with protein A-agarose overnight at 4°C
  • Non-IgGs were collected by loading the slurry into a column and washing with 2ml PBS.
  • IgGs were eluted with 2ml of 0.2M glycine pH 2.5 and neutralized in 200 ⁇ l Tris-HCl, pH
  • Protein samples were prepared for SDS-PAGE by diluting 1 : 1 with 2X denaturing buffer (40 ⁇ g/ml SDS, 20% glycerol, 30 ⁇ g/ml DTT and lO ⁇ g/ml bromophenol blue in 125mM Tris, pH 6.8), and boiling for 2min.
  • 2X denaturing buffer 40 ⁇ g/ml SDS, 20% glycerol, 30 ⁇ g/ml DTT and lO ⁇ g/ml bromophenol blue in 125mM Tris, pH 6.8
  • Pre-cast Nu-PAGE 10% Bis-Tris protein gels were obtained from Invitrogen (Carlsbad, CA). Each lane was loaded with a maximum of lO ⁇ l of sample, and run for 50min at 200 V.
  • the gel was stained with Invitrogen SafeStain (1 hour in staining solution, then washed overnight with water).
  • the GlycoTrack glycoprotein detection kit was obtained from Prozyme (formerly Glyko). All reagents except buffers were supplied with the kit. Two methods were attempted — either biotinylating glycoproteins after blotting (a) or before blotting (b). For both methods, samples were first diluted 1:1 with 200mM sodium acetate buffer, pH 5.5. The membrane was blocked by incubating overnight at 4°C with blocking reagent, then washed 3x10 minutes with TBS.
  • samples were denatured with SDS sample buffer, and subjected to SDS-PAGE and blotting to nitrocellulose.
  • the proteins were oxidized with 10ml of lOmM sodium periodate in the dark at room temperature for 20 minutes.
  • the membrane was washed 3 times with PBS, and 2 ⁇ l of biotin-hydrazide reagent was added in 10ml of lOOmM sodium acetate, 2mg/ml EDTA for 60 minutes at room temperature. After 3 washes with TBS, the membrane was blocked overnight at 4°C with blocking reagent.
  • S-AP streptavidin-alkaline phosphatase conjugate
  • TBS streptavidin-alkaline phosphatase
  • 50 ⁇ l of nitro blue tetrazolium (50mg/ml) and 37.5 ⁇ l of 5-bromo-4-chloro-3-indolyl phosphate /?-toluidine (50mg/ml) were added in 10ml TBS, lOmg/ml MgCl 2 . After 60 minutes, the blot was washed with distilled water and allowed to air dry.
  • PVDF-coated wells in a 96-well plate were washed with 200 ⁇ l MeOH, 3x 200 ⁇ l H 2 O and 200 ⁇ l RCM buffer (8M urea, 360mM Tris, 3.2mM EDTA, pH 8.3).
  • the protein samples (50 ⁇ l) were then loaded in the wells along with 300 ⁇ l RCM buffer.
  • 500 ⁇ l of 0.1M DTT in RCM buffer was added for lhr at 37°C. To remove the excess DTT, the wells were washed three times with H 2 O.
  • albumin Although the overwhelming amounts of albumin can obscure analysis for proteomics, it may not interfere with N-glycan profiling. There are also large amounts of glycosylated antibodies, which have a number of glycan structures [20, 21]. However, simple methods exist to separate these abundant antibodies from the less abundant glycoproteins. When working with serum, there are several issues to consider that are not relevant for single protein systems. Because the proteins in the sample are so concentrated, they can easily precipitate out of solution. Also, even though albumin does not have N-linked sugars, the sheer quantity present may interfere with glycan release or purification. There are several other major proteins in serum (i.e. immunoglobulins) that are N-glycosylated, which may overshadow the signals from less abundant proteins.
  • Identifying glycan structures with complex protein mixtures can be rather difficult.
  • all possible monosaccharide composition was assigned to each peak observed in a MALDI-MS spectrum.
  • each mass peak corresponds uniquely to a monosaccharide assignment.
  • Sample preparation Serum samples generally contained upwards of 120mg/ml of protein, making heat denaturing less ideal. Even when diluted, the proteins in these samples precipitated rapidly, giving the sample a gel-like consistency. This could prevent the PNGaseF from accessing all the N-glycan sites on the proteins.
  • One set of samples was processed using the traditional heat-denaturation method after diluting the serum samples 1 : 10 in water (Fig. 9A). A number of glycan peaks were observed in the MALDI-MS spectrum, but there clearly was residual detergent contamination from the denaturing step. On a separate sample, EndoF was used since the enzyme can act on folded proteins (Fig. 9B).
  • EndoF cleaves between the first and second GlcNAc on the glycan core, causing a loss of information on core fucosylation.
  • the samples were purified as usual.
  • glycans could indeed be obtained using both methods.
  • EndoF spectra had a relatively high level of baseline noise, and signal intensities were relatively low ( ⁇ 1000), leaving room for improvement.
  • the proteins were reduced with dithiothreitol (DTT) followed by carboxymethylation with iodoacetic acid to denature the proteins [22]. Reduction disrupts the disulfide bonds in proteins, while carboxymethylation prevents the proteins from re-folding.
  • DTT dithiothreitol
  • the neutral sugars were analyzed in positive ion mode in the MALDI-MS, while acidic sugars were examined in negative mode (Fig. 10).
  • the positive mode spectrum was checked for charged glycans.
  • This process was repeated multiple times with the same serum sample to ensure reproducibility (three aliquots were purified in parallel on one day, and another two on two different days).
  • multiple normal serum samples were processed by this method to determine the degree of glycan variation between serum samples.
  • Concanavalin A is a lectin that binds to ⁇ -linked mannose, as contained in all N-glycans [23].
  • a serum sample was passed through a column of agarose- bound ConA. Proteins containing N-glycans bound to the column while non-glycosylated proteins were washed off (this sample was collected as the ConA flow through). The glycoproteins were then eluted with a 500mM ⁇ -methyl-mannoside solution, which competes for the ConA binding sites.
  • the ConA flow through and elution samples were run on an SDS- PAGE gel (Fig. 12A). In the gel, the albumin fraction is clearly visible in the flow through from the ConA column, while multiple bands in the elution lane represent glycoproteins.
  • the glycan profiles of both the flow through and the elution fraction were analyzed. After dialyzing the samples against lOmM phosphate buffer, they were processed with PNGaseF and purified by C18 cartridge and GlycoH as described above. There were no observable glycans present in the flow-through fraction, while neutral and acidic sugars from the elution fraction are shown in Figs. 12B and 12C. The results from total serum digests, however, yield MALDI-MS data with signal-to-noise ratio and signal intensity that are as good as or better than from ConA elution. Therefore, in some cases there will be little to no advantage to removing non-glycosylated proteins before analysis.
  • Serum samples were also depleted of antibodies through a Protein A column to determine how many major peaks in the final spectra came from IgG.
  • the presence of glycoproteins in both the flow through and elution fractions were determined by GlycoTrack glycoprotein detection kit (Prozyme/Glyko) (Fig. 13A).
  • the Protein A elution fraction containing IgGs was treated with PNGaseF and purified as described above.
  • Figs. 13B-13E show a comparison of the glycans from IgG (Protein A elution) to the total glycan profile.
  • the three matrix preparations were 1) saturated DHB in water with 300mM spermine, 2) 20mg/ml DHB in acetonitrile and 25mM spermine in water in a 1 : 1 ratio and 3) 20mg/ml DHB in methanol and 25mM spermine in water in a 1 : 1 ratio.
  • Preparation 2 yielded MALDI-MS spectra with the highest signal-to-noise ratio in both positive and negative mode, and was used for all experiments. There are several reported methods for increasing the sensitivity and ionization efficiency of mass spectrometry data in the analysis of glycans.
  • glycan pools as a mixture of neutral and acidic glycans, as the chemical properties of the glycans are modified to allow for more uniform ionization.
  • Two types of chemical modifications were tested to determine whether the MALDI-MS results could be improved upon.
  • N-glycan samples are commonly permethylated to protect each OH and ⁇ H 2 or amide group in the carbohydrate [24]. This is particularly useful for MS techniques such as fast atom bombardment (FAB-MS), since permethylated glycans fragment in a much more predictable manner than underivatized glycans. Permethylation can also increase sensitivity in electrospray (ES-MS) and MALDI-MS.
  • FAB-MS fast atom bombardment
  • Fig. 14 The schematic of the permethylation reaction is shown in Fig. 14.
  • Some drawbacks to permethylation are that the sample has to be extremely clean for the reaction to go to completion, and the sample requires clean-up after the reaction.
  • this method slightly improved the ionization of N-glycan standards in MALDI-MS over non-modified glycans, the increase in signal-to-noise ratio was not significant (Fig. 15).
  • a newer method for increasing N-glycan ionization, as well as allowing the glycans to ionize more uniformly across species is to conjugate it to a peptide [25].
  • the structure of the peptide and its glycan conjugation reaction are shown in Fig. 16.
  • the peptide' s active hydroxylamine group readily reacts with any aldehydes or ketones present, thus preventing it from conjugating to the glycans.
  • the reaction with glycan standards displayed promising results, it was difficult to obtain a complete reaction with serum samples. Even after several attempts to label serum glycans with varying amounts of peptide, free glycan peaks in the spectra were observed from flow-through and water wash spots. Because there may be excess aldehydes or ketones remaining in serum samples, peptide conjugation was not used, and the samples were analyzed as separate neutral and acidic fractions.
  • the sample was dialyzed against phosphate buffer, pH 7.5 or Tris acetate pH 8.3 overnight and concentrated to ⁇ 200 ⁇ l in a spin column with a 3000Da MWCO filter. To cleave the sugars from the protein between 100 and 2,000U of PNGaseF (New England Biolabs, Beverly, MA) were used. C) Glycoproteins were denatured using a buffer containing 8M urea, 3.2 mM EDTA and 360 M Tris, pH 8.6 [Papac, 1998]. Reduction and carboxymethylation of the glycoproteins was then achieved using DTT and iodoacetic acid (or iodoacetamide), respectively.
  • N-glycans were selectively released from the glycoproteins by incubation with P ⁇ Gase F.
  • Glycan samples were loaded onto the column in water, and washed through with 3ml H 2 O. The flow through was collected and lyophilized to obtain the desalted sugars.
  • D) GlycoClean S cartridges (Prozyme, San Leandro, CA; formerly Glyko), were primed with 1ml water and 1ml 30% acetic acid, followed by 1ml acetonitrile.
  • the glycan sample was loaded (in a maximum volume of lO ⁇ l) onto the disc, and the glycans were allowed to adsorb for 15 minutes. After washing the disc with 1ml of 100% acetonitrile and 5 x lml of 96% acetonitrile, glycans were eluted with 3 x 0.5ml water.
  • GlycoClean H cartridges (Prozyme; 200mg bed) were washed with 3ml of IM ⁇ aOH, 3ml H 2 O, 3ml 30% acetic acid, and 3ml H 2 O to remove impurities.
  • the matrix was primed with 3ml 50% acetonitrile with 0.1% TFA (Solvent A) followed by 3ml 5% acetonitrile with 0.1% TFA (Solvent B). After loading the sample in water, the column was washed with 3ml H 2 O and 3ml Solvent B. Finally, the sugars were eluted using 4x0.5ml of Solvent A. GlycoClean H cartridges can be reused after washing with 100% acetonitrile and re-priming with 3ml of Solvent A followed by 3ml of Solvent B. For the 25mg cartridge, wash volumes were reduced to 0.5ml.
  • N-Glycans Chemical Modification of N-Glycans
  • derivatization methods are currently used to increase the sensitivity and ionization efficiency of mass spectrometry data in the analysis of glycans. With these methods, it is often possible to analyze glycan pools as a mixture of neutral and acidic glycans, as the chemical properties of the glycans are modified to allow for more uniform ionization.
  • N-glycan samples are commonly permethylated to protect each OH and NH 2 or amide group in the carbohydrate. This is particularly useful for MS techniques such as fast atom bombardment (FAB-MS), since permethylated glycans fragment in a more predictable manner than underivatized glycans.
  • FAB-MS fast atom bombardment
  • Permethylation can also increase sensitivity in electrospray (ES-MS) and MALDI-MS.
  • ES-MS electrospray
  • MALDI-MS Permethylation
  • glycans in water were placed in a round-bottomed flask and lyophilized overnight.
  • a slurry of NaOH in DMSO (0.5ml) was added to the glycan sample, along with 0.5ml methyl iodide and incubated for 15 minutes.
  • the sample was then diluted with water and extracted 2X with CHC1 3 , collecting the organic phase. After drying the organic phase with MgSO , it was filtered through glass wool and dried under vacuum. Samples were then redissolved in methanol for MALDI-MS analysis.
  • glycopeptides were purified by C18, 0.6 ⁇ l bed ZipTip (Millipore, Billerica, MA). Specifically, the tip was washed with 5 ⁇ l of 100% acetonitrile, followed by water and 5% acetonitrile, 0.1% TFA. To load the sample, 2 ⁇ l of sample was drawn into the tip, and discarded after 5 seconds. After washing 3X with 5 ⁇ l H 2 O, glycopeptides were eluted with 10% acetonitrile.
  • the peptide' s hydroxylamine group readily reacts with any aldehydes or ketones present, thus preventing it from conjugating to the glycans.
  • Other labeling reagents i.e. APTS, ANTS, AMAC, etc.
  • APTS APTS, ANTS, AMAC, etc.
  • the matrix for glycans was utilized in combination with spermine (20mg/ml DHB in acetonitrile and 25mM spermine in water in a 1 :1 ratio.).
  • This recipe resulted in detection limits of 1 pmol and 10 pmol for neutrals and acidic glycan respectively.
  • Significant peak splitting with multiple sodium and potassium ions were observed.
  • this matrix crystallized as long needle-shaped crystals, which makes it difficult to achieve reproducible quantification of glycans present in a sample and eliminates the possibility for the automation of data acquisition.
  • Some of the matrices and reagents used in this study were: cafeic acid, dihydroxybenzoic acid (DHB), spermine, 1-hydroxyisoquinoline (HIQ), 6-aza-2-thiothymine (ATT), 2,4,6-trihydroxyacetophenone (THAP), Nafion, 6-hydroxypicolinic acid, 3- hydroxypicolinic, 5-methoxysalicylic acid (5-MS A), ammonium citrate, ammonium tartrate, sodium chloride, ammonium resins, etc.
  • These reagents were used in combination with different solvents such as methanol, ethanol, acetonitrile and water.
  • the matrix of matrices study resulted in new recipes of 2,5-dihydroxybenzoic acid (5 mg/ml) and 5-methoxysalicylic acid (0.25 mg/ml) in acetonitrile afor neutrals and 6-aza-thiothymine (10 mg/ml in Ethanol) spotted on Nafion coating for acidic glycans.
  • These matrices displayed the best detection limits for a mixture of carbohydrates to our knowledge: 25 fmol and 5 ftnol for neutrals and acidic glycans respectively (Fig.20).
  • the new matrices also showed minimum peak splitting, highly uniform signal intensity, spot morphology and no detectable fragmentation.
  • the dynamic range of these matrices were in the low fmol range ensuring that changes in low abundant glycans can be accurately monitored by using these matrices.
  • three methods were used. For the crushed spot method, l ⁇ l of matrix was spotted on the stainless steel MALDI-MS sample plate and allowed to dry. After crushing the spot with a glass slide, l ⁇ l of matrix mixed 1:1 with sample was spotted on the seed crystals and allowed to dry. Alternatively, 1 ⁇ l of matrix was applied followed by 1 ⁇ l of sample, or vice versa.
  • LC-MS LC-MS/MS
  • CE-LIF electrospray ionization mass spectrometer
  • CE-LIF capillary electrophoresis-laser induced fluorescence
  • the carbohydrates are first derivatized, in some preferred embodiments, by a reductive amination, at their reducing end with a fluorescent molecule such as APTS, ANTS, AMAC, etc.
  • the fluorescently-modified (or "labeled") carbohydrates are then separated by capillary electrophoresis and detected with high sensitivity via laser induced fluorescence.
  • glycosidases can also used in combination with CE-LIF in order to get further structural linkage information on the carbohydrates.
  • Step 1 Separate the glycans from the glycopeptide mixture. Isolate and sequence the resultant peptide(s). In this example, there was only one peptide chain and that was determined to be - YCNISQKMMSRNLTKDR. This peptide has two possible N glycosylation sites: C S and RNLT.
  • the peptide fragments with mass 475 and 855 contain the glycoslylation sites - both glycoslyation sites are glycosylated.
  • YCNISQKMMSRNLTKDR trypsin digest simulation of the peptide
  • the Asn residue is converted into an Asp residue which results in a total increase in molecular weight of IDa, thus explaining the appearance of the 476 and 856 peaks.
  • the deglycosylation with concomitant ls O-labeling results in an increase of 2Da in the peptides that originally had a glycosylation site. This explains the appearance of the 478 and 858 peaks.
  • the quantitative measurement of the peaks via the methods described above reveals that the glycosylation site at NLTK is 75% glycosylated. Similarly, the data for YCNISQK reveals that it is 50% glycosylated. Similarly the undigested glycopeptide mixture is also cleaved of the glycans and label processed as described above. The resultant analysis shows that the entire mixture is 75% glycosylated.
  • Step 4 Digest the glycopeptide mixture with trypsin and analyze the resultant mixture through MALDI-MS.
  • the resultant masses are 289, 475, 523, 854, 1871, 2033 and 2089.
  • Fragment NLTK is glycosylated with glycans with mass 1397 and 1559.
  • Fragment YCNISQK is glycosylated with glycan with mass 1235.
  • o Chain A that is not glycosylated.
  • o Chain B in which the second Asn is glycosylated with Glycan- 1397.
  • o Chain C in which the second Asn is glycosylated with Glycan - 1559.
  • o Chain D in which the first Asn is glycosylated with Glycan 1235.
  • o Chain E in which the first Asn is glycosylated with 1235 and the second with 1397.
  • o Chain F in which the first Asn is glycosylated with 1235 and the second with 1559.
  • Step 5 Generate equations based on the experimental results and/or other data.
  • a,b,c,d,e and f are the relative abundances of the chains A, B, C, D, E and F respectively, and the following set of equations were generated based on the experimental results from steps 1 through 4.
  • Step 6 The masses from step 3 can be resolved into potential glycan structures by using a glycan database lookup (http://www.functionalglycomics.org/glycomics/molecule/jsp/carbohydrate/searchByMw.jsp), and the exact structure of the carbohydrates were corroborated from the glycosidase digest analysis. By putting together the results in steps 1 to 6, the unknown glycoprotein mixture was determined to be (Tables 9 and 10):
  • the glycoproteins are first denatured in the presence of urea, reduced with DTT and carboxymethylated with iodoacetamide. To remove the denaturing reagents, the samples are concentrated using a centrifugal concentrator (3,000 MWCO) followed by buffer exchanged into protease compatible buffer (50 mM ammonium bicarbonate, pH 8.5, for trypsin digest). The proteins are then cleaved by proteases followed by denaturation of proteases by boiling the sample in water and liophilization.
  • protease compatible buffer 50 mM ammonium bicarbonate, pH 8.5, for trypsin digest
  • Glycosylation site specific labeling is achieved by reacting the samples with PNGase F in the presence of 18 O-water (Fig. 24). After desalting the glycosylated, unglycosylated, and 18 O-labeled unglycosylated peptides through a C-18 solid phase extraction cartridge, these are used for LC-MS, LC-MS/MS, MALDI or MALDI-FTMS. For this study the 16 O and 18 O labeled samples were mixed in a 1 : 1 ratio before injection in order to facilitate the analysis. Other techniques for peptide sequencing can also be used at this point.
  • the peptides were analyzed using a capillary LC-MS using a Vydac C-18 MS 5 ⁇ m (250x0.3mm) column coupled to a Mariner Biospectrometry Workstation.
  • the peptides generated from the protease cleavage were corroborated using the Swiss-Prot database (ribonuclease B, P00656 and ovalbumin, P01012).
  • the specific glycosylation site can be easily determined.
  • the introduction of the 18 O at the glycosylation site is detected as a 2Da increase for a specific peptide. This data facilitate the determination of the glycosylation site and its occupancy.
  • the tryptic digest of ribonuclease B should yield a peptide fragment with a
  • N-glycanase F PNGaseF was chosen among enzymatic methods for the cleavage of N-linked glycans since the use of other enzymes results in loss of information such as fucosylation at the proximal GlcNAc.
  • proteins were unfolded, reduced and carboxymethylated prior to enzymatic digestion.
  • the samples were denatured by heating in the presence of ⁇ -mercaptoethanol and/or SDS or by incubating at room temperature with urea, followed by reduction with DTT and carboxymethylation with iodoactic acid or iodoacetamide.
  • the proteins were first precipitated with ethanol and the supernatant containing the glycans was then dried under vacuum and resuspended in water. Subsequent purification steps were required when detergents were used.
  • Optimal results were obtained by using porous graphitic carbon columns. Neutral and charged carbohydrates were separated using these columns and eluted in mass spectrometry-compatible buffers. At this point, the most difficult component to get rid of was the detergent, which interferes with the types of analytical techniques that were used in this study.
  • Fig. 26 shows the MALDI-MS spectrum of ovalbumin glycans. The observed peaks and their structures were found. The results are as shown above in Table 3.
  • RNAse B Computational analysis The information obtained from the previous analysis was analyzed using the computational platform that contains the proteomics and glycomics based bioinformatics tools and databases described herein.
  • the sequence of the protein backbone was determined from the proteomics database as follows:
  • glycosylation site is at SNLT. It is 100 % glycosylated and five different glycans were observed on analysis of the glycans via MALDI-MS. The results of the computational analysis showed that there were 5 different chains in the glycoprotein mixture as shown in Table 11 below:
  • pH can be controlled automatically by the instrument, which dispenses CO 2 , NaHCO 3 and O 2 as needed.
  • measurements must be taken manually and pH adjusted by hand.
  • the pH in this reactor can be controlled by either adding fresh media as the cells grow, or adding NaHCO 3 for increased buffering capacity, and CO 2 as needed.
  • the main difference between the reactor types is the mode of agitation.
  • a blade stirrer keeps the cell suspension in motion, while a sparger introduces oxygen to the system in a controlled manner.
  • a rocking motion generates waves that mix the components of the system and aids the transfer of oxygen and other gases into the system.
  • the purified antibodies were processed according to the optimized method described above.
  • CD22 a B cell- specific immunoglobulin superfamily member, is a sialic acid-binding lectin. JBiol Chem 268, 7011-8.
  • Sample preparation and carbohydrate purification Samples (usually 60 ⁇ l) from the different body fluids (serum, saliva, urine, tears, etc.) were processed in a similar manner as described below. Although in most cases the entire glycoproteome from the sample was analyzed, in some cases, the samples were further fractionated in order to analyze a "sub-glycome" from a specific body fluid. For example, a specific subset of proteins (such as antibodies, serum albumins, and other high abundance proteins) were removed from the original serum sample in order to analyze a more specific subset of glycoproteins in more detail. For fractionation, the sample proteome was divided into "high abundance” and "low abundance” using solid supports containing antibodies, proteins and synthetic molecules specific for the desired proteins to be removed or concentrated.
  • proteins such as antibodies, serum albumins, and other high abundance proteins
  • IgGs were removed using protein A agarose (Biorad), beads and serum albumin was removed using Affi-blue gel (Biorad).
  • Other fractionations included the separation into acidic and basic proteome using cation and anion exchange chromatography or the separation between glycosylated and unglycosylated proteins using Con- A columns. The removal of specific proteins was quantified by western blots. Proteins in the samples (either fractionated or unfractionated) were then denatured using a buffer containing 8M urea, 3.2 mM EDTA and 360 mM Tris, pH 8.6.[Papac, 1998]. Reduction and carboxymethylation of the sample proteome was then achieved using DTT and iodoacetamide respectively.
  • N-glycans were selectively released from the glycoproteins by incubation with P ⁇ Gase F.
  • the steps for protein denaturing, protein alkylation and glycan release were also performed with the proteins bound to a solid support as described below.
  • the released carbohydrates were then purified from the proteins and separated into neutrals and acidic glycans in one step using a graphitized carbon columns.
  • the glycan purification step was also performed in a high-throughput format by using columns in 96-well plates. This process was facilitated by the use of a TECA ⁇ robot. This protocol allowed the processing of more than 90 samples at the same time.
  • Affi-Blue Gel Biorad, 200 ⁇ L
  • Prot A Biorad, 200 ⁇ L
  • the column was washed with lmL of compatible serum protein binding buffer (20 mM phosphate, 100 mM ⁇ aCl, pH 7.2) using gravity flow.
  • the column was placed in an empty 2mL collection tube and centrifuged at 10,000G for 20 seconds at 4°C The flow was stopped during the sample preparation.
  • Serum 60 ⁇ L was mixed with compatible serum protein binding buffer (180 ⁇ L), and 200 ⁇ L of diluted serum was added to the top of the resin bed and allowed to mix with the column for 15 minutes. The column was then centrifuged at 10,000G for 20 seconds at 4C Using the same collection tube, the column was washed with 200 ⁇ L of compatible serum protein binding buffer and centrifuged again at 10,000G for 20 seconds at 4°C For the removal of IgGs alone, only protein A agarose beads were used and the binding buffer was modified to 10 mM phosphate, 150 mM ⁇ aCl, pH 8.2.
  • Concanavalin A-agarose beads (Vector Laboratories, Burlingame, CA) were used. To prepare the column, 3ml ConA-agarose slurry was washed with ConA buffer (20mM Tris, lmM MgCl 2 , ImM CaCl 2 , 500mM ⁇ aCl, pH 7.4). Before loading, 500 ⁇ l of serum was mixed with 150 ⁇ l of 5X ConA buffer.
  • Lane one contained 5ul of a standard (BioRad: Precision All Blue Standard, 161-0373). The gel was run for 70 minutes at 200V. The gel was stained with SimplyBlue (Invtrogen:LC6060) according to the manufacturer. Imaging was performed on a Kodak Image Station 2000R. Another set of duplicate depleted samples were run as before and one gel was for SimplyBlue and the other was transferred to a 0.20um nitrocellulose membrane (Invitrogen:LC2000) employing an X Cell Blot Module (Invitrogen:E19051) for 70 minutes at 30V.
  • SimplyBlue Invtrogen:LC6060
  • Another set of duplicate depleted samples were run as before and one gel was for SimplyBlue and the other was transferred to a 0.20um nitrocellulose membrane (Invitrogen:LC2000) employing an X Cell Blot Module (Invitrogen:E19051) for 70 minutes at 30V.
  • the membrane was then blocked overnight at 4°C in 5% Blotto (Santa Cruz:sc- 2325) and then probed with 1:1000 Protein A-hrp (Zymed :10-1023) for 1 hour at 4°C and washed 4 times with washing buffer (lxTBS:200mMTris base, 1.5M NaCl, pH7.5).
  • the blot was developed with 4ml of substrate (ECL plus Western Blotting Detection System:RPN2132) for 2 minutes and then exposed.
  • the bands corresponding to the treatments were manually captured as ROI (region of interest) employing the Kodak ID Image Analysis Software and the Mean Intensity was normalized to the controls.
  • the membrane was washed 3 times with PBS, and 2 ⁇ l of biotin-hydrazide reagent was added in 10ml of lOOmM sodium acetate, 2mg/ml EDTA for 60 minutes at room temperature. After 3 washes with TBS, the membrane was blocked overnight at 4°C with blocking reagent. Before adding 5 ⁇ l of streptavidin-alkaline phosphatase (S-AP) conjugate, the membrane was washed again with TBS. The S-AP was allowed to incubate for 60 minutes at room temperature, and excess was washed off with TBS.
  • S-AP streptavidin-alkaline phosphatase
  • nitro blue tetrazolium 50 ⁇ l of nitro blue tetrazolium (50mg/ml) and 37.5 ⁇ l of 5-bromo-4-chloro-3-indolyl phosphate p-toluidine (50mg/m ⁇ ) were added in 10ml TBS, lOmg/ml MgCl 2 . After 60 minutes, the blot was washed with distilled water and allowed to air dry. In method (b), 20 ⁇ l of sample was mixed with 1 O ⁇ l of 1 OmM periodate in 1 OOmM sodium acetate, 2mg/ml EDTA and incubated in the dark at room temperature for 20 minutes.
  • Glycome analysis using mass spectrometry Glycan analysis using methods known to the art were used and applied to the total body fluid glycome analysis. Using the methods provided above were also able to analyze more than 90 samples. Optimized MALDI-MS methods which did not required additional labeling and purification steps and also displayed great reproducibility and sensitivity for the carbohydrate analysis was used. As shown in Fig.31, total serum glycome profiles typically displayed between 25-30 neutral glycans as well as 25-30 acidic glycans. Using the look-up table described previously, almost all the peaks in MALDI-MS serum profiles could be identified as glycans of known composition. Many of the unidentified peaks are sodium adducts.
  • composition and mass of each labeled peak are as listed above in Table 18. However, a few peaks in the acidic glycan spectrum correspond to more than one composition. This is more common in the higher mass range since there are a larger number of possible monosaccharide compositions.
  • MALDI-MS analysis allows us to analyze the entire glycome profile in a sample and compare the changes in the glycome composition between samples in a rapid and efficient mariner. Due to the limitations in isomass characterization, in some instances, other techniques known to the art can be used to further characterize and validate the biomarkers determined from the total profile found using MALDI-MS techniques. For example, liquid chromatography-mass spectrometry (LC-MS) and capillary electrophoresis-laser induced fluorescence (CE-LIF), are used in combination with a panel of exoglycosidases in order to obtain further linkage characterization of the carbohydrates (Fig. 32).
  • LC-MS liquid chromatography-mass spectrometry
  • CE-LIF capillary electrophoresis-laser induced fluorescence
  • LC-MS/MS is also used to obtain linkage information based on the fragmentation patterns.
  • Glycome analysis of cell surface glycoproteins The methods are also applied to the glycoprofiling of cell surfaces. All cell surface glycoproteins are cleaved using methods know to the art. Briefly, to harvest glycans using protease extraction, cells are washed 3X with PBS and incubated for 20-45 minutes with trypsin/EDTA (GibcoBRL) at 37°C for protease extraction. The samples are centrifuged for 10 minutes at 3000xg to pellet the cells, and the supernatant containing glycopeptides is collected and processed using methods described herein.
  • proteomic pattern diagnostics have been adapted and applied to total glycomic pattern analysis where the total profile of carbohydrates from body fluids or tissues can be examined in a rapid format.
  • This approach provides an efficient overview of the total changes in carbohydrate composition of a tissue or body fluid as a result of pathological alterations and should be very reliable in sensing susceptible physiological changes to the body's natural homeostasis.
  • This method not only serves as a fast diagnostic/prognostic tool but should also help to understand the function of specific carbohydrate modifications in some diseases. This method also provides a reliable system to efficiently monitor the effects of therapeutics.
  • the optimization of the MALDI-MS analysis allows reliable reproducibility that enables the fast evaluation of alterations to the glycomic patterns and their subsequent association to pathological/physiological changes to a sample donor.
  • the optimized detection limits for this method (low femtomol) allows the detection of low abundance species associated to diseases. Every signal in the pattern is rapidly correlated to the glycan identity and can be further validated using a panel of glycosydases and other techniques. This prevents the erroneous identification as it has sometimes been the case in the field of proteomic pattern diagnostics.
  • the pattern alterations can be easily determined manually or more efficiently with the aid of bioinformatics tools (described below). In some cases the decreasing levels of circulating glycoproteins in serum are easily matched to the analyzed glycans.
  • the glycome profile from serum with low IgG levels reflects the specific decrease in the respective IgG glycans with molecular weights of 1463, 1626, 1666, 1788, 1829, 2102, and 1844. These glycans have been previously shown to be attached to IgG molecules in serum [Butler, 2003].
  • specific alterations in the glycomic pattern that can be correlated to the pathological state of the donor can be determined. For example, glycomic patterns have been associated to prostate cancer by studying the serum from prostate cancer patients (Fig. 35). Glycomic patterns from the saliva of patients with viral infections have also been established.
  • These features can be the presence or absence of one or more glycans in the profile, the relative amount of different glycans in the profile, combinations of different glycans found in the profile and other glycan-related properties.
  • These glycans are identified in the glycoprofile spectra and can be corroborated with other methods, for instance, by using a Glycan database (http://www.functionalglycomics.org/glycomics/molecule/jsp/carbohydrate/carbMoleculeHo me.jsp) and/or associated glycomics-based bioinformatics tools.
  • the appropriate patient population is selected for the study (based on their history in a patient database), such that the subjects chosen in the different categories of prostate cancer, BPH and normal have the same distribution when it comes to other properties such as age, ethnicity, behavioral factors etc. This ensures that the variation in the glycan profiles can be attributed to the disease condition rather than other factors.
  • the glycan related features extracted for this population via the previous step is run through a dataset generator to create the datasets needed for pattern analysis [see Weiss, S. & Indurkhya, N. Predictive data mining - A practical guide, (Morgan Kaufmann, San Francisco, 1998)]. Different types of pattern analysis are performed to identify the patterns in this dataset
  • d j can be Prostate Cancer, BPH or Normal o Decision Rules:
  • the pattern identified is in the form of IF-THEN rules, for example (IF Gii is present and G 7 is not present) or (W G 8 is present and Gg is present)
  • THEN Class Prostate Cancer QF Gi is present and G 2 is present and G 3 is not present)
  • the patterns, rules or relationships are validated.
  • the validation can be made based on variety of statistical methods that are used in biomarker validation as well as scientific methods to verify that the glycans found in the patterns do accurately reflect the disease state. If the patterns cannot be validated, the process described above can be repeated to look for other glycan-based patterns in the glycoprofiles.

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Abstract

L'invention se rapporte entre autres, à une analyse améliorée de glucides. Elle se rapporte en particulier à l'analyse de glucides, du type N-glycanes et O-glycanes trouvés sur des protéines. L'invention se rapporte en outre à des procédés améliorés permettant l'étude de motifs de glycosylation sur des cellules, des tissus et des fluides corporels. Les informations relatives à l'analyse de glycanes, telles que les motifs de glycosylation sur des cellules, des tissus et dans des fluides biologiques peuvent être utilisées dans des procédés de diagnostic et de traitement ainsi que pour faciliter l'étude des effets d'une glycosylation/glycosylation modifiée sur une fonction de protéine. L'invention se rapporte également à ces procédés. Elle se rapporte en outre à des procédés permettant d'évaluer les processus de production de protéines, qui permettent d'évaluer la pureté des protéines produites, ainsi que pour sélectionner des protéines avec la glycosylation désirée.
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